U.S. patent application number 15/435438 was filed with the patent office on 2017-08-24 for method for the immobilization of biomolecules.
The applicant listed for this patent is Boehringer Ingelheim Vetmedica GmbH. Invention is credited to Matthias GRIESSNER, Ralf KRAEHMER, Frank LEENDERS, Heinz SCHOEDER.
Application Number | 20170241998 15/435438 |
Document ID | / |
Family ID | 55405244 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170241998 |
Kind Code |
A1 |
SCHOEDER; Heinz ; et
al. |
August 24, 2017 |
METHOD FOR THE IMMOBILIZATION OF BIOMOLECULES
Abstract
The invention relates to a method for the immobilization of
biomolecules containing at least one sulfhydryl group, which method
comprises contacting a modified metal surface with the biomolecule
irradiating the resulting surface with UV radiation in the presence
of a photo-initiator, wherein said metal surface is modified with a
cross-linker compound comprising a terminal thiol or dithiol group
covalently linked to the metal surface, a spacer group, which at
the other terminal end is carrying an isolated double or triple
bond.
Inventors: |
SCHOEDER; Heinz;
(Isernhagen, DE) ; GRIESSNER; Matthias; (Hannover,
DE) ; LEENDERS; Frank; (Berlin, DE) ;
KRAEHMER; Ralf; (Panketal, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim Vetmedica GmbH |
Ingelheim am Rhein |
|
DE |
|
|
Family ID: |
55405244 |
Appl. No.: |
15/435438 |
Filed: |
February 17, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 17/14 20130101;
G01N 33/54393 20130101; C07K 1/1077 20130101; G01N 33/553 20130101;
G01N 33/54353 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C07K 1/107 20060101 C07K001/107; G01N 33/553 20060101
G01N033/553; C07K 17/14 20060101 C07K017/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2016 |
EP |
16156777.1 |
Claims
1. A method for the immobilization of biomolecules containing at
least one sulfhydryl group, which method comprises the steps of: a)
optionally treating a biomolecule with an reducing agent in order
to cleave existing --S--S-- bridges in the biomolecule, or b)
optionally treating a biomolecule with an acylation agent carrying
a protected sulfhydryl group and de-protecting the sulfhydryl
group; c) contacting a modified metal surface with the biomolecule;
d) irradiating the resulting surface with UV radiation in the
presence of a photo-initiator, wherein said metal surface is
modified with a cross-linker compound comprising: i) a terminal
thiol or dithiol group covalently linked to the metal surface being
connected to ii) a spacer group, which at the other terminal end is
carrying iii) an isolated double or triple bond.
2. A method according to claim 1, wherein the linker compound is a
compound of formula (I),
HS--(CH.sub.2).sub.m--CH(ZH)-SPACER-(CH.sub.2).sub.p-A (I) in which
m is an integer from 2 to 6, A is selected from --CH.dbd.CH.sub.2
and --C.ident.CH, and Z is S or a single bond, SPACER is a group of
formula
--(CH.sub.2).sub.n--(C.dbd.O).sub.x--Y--(CH.sub.2CH.sub.2--O).sub.y--(CH.-
sub.2).sub.r--(C.dbd.O).sub.v--X-- wherein X and Y are each
independently NH or O, n is 0 or an integer from 1 to 10, x and v
are each independently 0 or 1, y is an integer from 1 to 20, r and
p are each independently selected from an integer from 1 to 6.
3. A method according to claim 2, wherein Z is S, A is
--CH.dbd.CH.sub.2, and m is an integer from 2 to 4, X and Y are NH,
n is 0 or an integer from 1 to 10, x and v are 1, y is an integer
from 1 to 20, r and p are 1.
4. A method according to claim 1, wherein the biomolecule is an
antibody, an enzyme or nucleic acid.
5. A method according to claim 1, wherein the photo-initiator is a
1-benzoyl-1-methyl-ethanol derivative.
6. A method according to claim 1, wherein the irradiation is
carried out at a wavelength .lamda..sub.max of 300 to 340 nm.
7. A cross-linker compound of formula (I),
HS--(CH.sub.2).sub.m--CH(ZH)-SPACER-(CH.sub.2).sub.p-A (I) in which
m is an integer from 2 to 6, A is selected from --CH.dbd.CH.sub.2
and --C.ident.CH, and Z is S or a single bond, SPACER is a group of
formula
--(CH.sub.2).sub.n--(C.dbd.O).sub.x--Y--(CH.sub.2CH.sub.2--O).sub.y--(CH.-
sub.2).sub.r--(C.dbd.O).sub.v--X-- wherein X and Y are each
independently NH or 0, n is 0 or an integer from 1 to 10, x and v
are each independently 0 or 1, y is an integer from 1 to 20, r and
p are each independently selected from an integer from 1 to 6.
8. A cross-linker compound of formula (IA),
HS--(CH.sub.2).sub.m--CH(SH)-SPACER-(CH.sub.2).sub.p-A (IA) in
which A, SPACER, m and p have the meaning given for formula (I) of
claim 7.
9. A cross-linker compound of formula (IA) according to claim 8,
wherein m is an integer from 2 to 4, p is 1. SPACER is a group of
formula
--(CH.sub.2).sub.n--(C.dbd.O)--NH--(CH.sub.2CH.sub.2--O).sub.y--CH.sub.2--
-(C.dbd.O)--NH-- in which n is 0 or an integer from 1 to 10, y is
an integer from 1 to 20.
10. An intermediate of formula (II) f, ##STR00014## wherein A,
SPACER, m and p have the meaning given for formula (I) of claim
7.
11. A modified metal surface in which at least one thiol group of
the compound of formula (I) in accordance with claim 7 is
covalently linked to at least one of the metal atoms of the
surface.
12. A modified metal surface according to claim 11 wherein the
metal is a noble metal, preferably gold.
13. A kit for carrying out a method of immobilizing biomolecules,
in accordance with claim 1, said kit comprising i) a substrate with
a modified metal surface in accordance with claim 11, ii) an
optional containment unit containing a suitable reducing agent, or
iii) an optional containment unit containing a suitable acylation
agent carrying a protected sulfhydryl group and a de-protection
agent for the sulfhydryl group iv) a containment unit containing a
suitable photo-initiator, and v) a leaflet explaining the
conditions for carrying out the method.
14. A kit for carrying out a method of immobilizing biomolecules
according to claim 13, which further comprises a suitable apparatus
for UV irradiation.
15. A modified metal surface in which at least one thiol group of
the compound of formula (IA) in accordance with claim 8 is
covalently linked to at least one of the metal atoms of the
surface.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The invention relates to a method for the immobilization of
biomolecules containing at least one sulfhydryl group, which method
comprises contacting a modified metal surface with the biomolecule
irradiating the resulting surface with UV radiation in the presence
of a photo-initiator, wherein said metal surface is modified with a
cross-linker compound comprising a terminal thiol or dithiol group
covalently linked to the metal surface, a spacer group, which at
the other terminal end is carrying an isolated double or triple
bond.
[0003] B. Description of the Related Art
[0004] Detection and quantification of analytes, such as
biomolecules or other molecules that affect biological processes,
present in samples are integral to analytical testing. For example,
the detection of biomolecules that are markers of biological
activity or disease is important for the diagnosis of medical
conditions and pathologies. However, converting the detection of an
analyte, such as a biomolecule, into a usable signal is challenging
in part due to the complexity of transducing the detection event,
for example antibodies binding an antigen, into a detectable signal
that can be converted into perceivable data. Some assays, such as
enzyme linked immunoabsorbant assays (ELISA) detect biomolecules by
monitoring the binding event which generates light or a reaction
product that produces a color change in the sample. One advantage
of these types of assays is that they are very sensitive. However,
a drawback of these assays, such as an ELISA assay, is that they
typically require long period of time to develop a detectable
signal and require multiple steps to complete.
[0005] Recently, other methods have been being developed that
retain the sensitivity of traditional immunoassays, while
eliminating the complexity and time involved in developing the
signal. One strategy is to couple the sensitivity of the
immunoassay, for example by using highly selective antibodies that
have high affinity for analytes, with electrochemical measurements.
By combining the detection events to an electric signal, the
information about the presence and concentration of an analyte in a
sample can be immediately converted to an electrical signal. Over
the past decades several sensing concepts and related devices have
been developed. The most common traditional techniques include
cyclic voltammetry, chronoamperometry, chronopotentiometry, and
impedance spectroscopy.
[0006] However, the general performance of electrochemical sensors
is often determined by the surface architectures that connect the
sensing element to the biological sample at the nanometer scale.
Electrochemical biosensors have suffered from a lack of surface
architectures allowing high enough sensitivity and unique
identification of the response with the desired biochemical
event.
[0007] Thus, the need exists for electrochemical bio sensors that
have the high sensitivity of traditional assays, such as ELISA
assays, while maintaining the desirable aspects of an
electrochemical sensor, such as readily measurable signal and the
prospects of miniaturization.
[0008] The US patent application US 2012/0228155 relates to a
method of making a functionalized electrode for detecting a target
analyte, comprising: contacting an electrically conducting surface,
e.g. a gold electrode with a mixture comprising a first thiol
compound having a terminal amino group and a second thiol compound
having a terminal OH, an alkoxy, a methyl, a sugar, a
zwitter-ionic, or a polar non-ionic group, wherein sulfhydryl
groups on the first and second thiol compounds bond with the
electrically conducting surface, thereby creating a monolayer on
the surface of the electrically conducting surface; contacting the
monolayer on the surface of the electrically conducting surface
with a hetero-bifunctional linker that comprises an amine reactive
functionality, and a diazirine or maleimide moiety; and contacting
the monolayer on the surface of the electrically conducting surface
with a ligand that specifically binds a target analyte, thereby
making a functionalized electrode for detecting a target
analyte.
[0009] If the hetero-bifunctional linker comprises sulfo-NHS
diazirine (sulfo-SDA), the methods further comprises exposing the
monolayer on the surface of the electrically conducting surface to
UV radiation, thereby making a functionalized electrode for
detecting a target analyte. The U.S. Pat. No. 8,580,571 relates to
a method for producing a biosensor comprising a substrate to which
a hydrophilic polymer is being bound, the method comprising the
following steps: forming a self-assembled monolayer on a substrate,
wherein the self-assembled monolayer is formed by an alkanethiol;
coating a solution containing a photo radical generator onto this
substrate to allow the photo radical generator to bind to the
self-assembled mono-layer on the substrate, coating a solution
containing a hydrophilic polymer onto this substrate, wherein the
hydrophilic polymer is a polysaccharide having a carboxyl group and
a double bond and exposing this substrate to light to generate a
reactive group from the photo radical generator and to covalently
bind the hydrophilic polymer to said reactive group via the double
bond of the hydrophilic polymer, whereby the biosensor comprising a
substrate to which a hydrophilic polymer is being bound is
produced, wherein the carboxyl group contained in the hydrophilic
polymer bound to the substrate in the biosensor is used for
immobilizing a physiologically active substance of interest onto
the biosensor.
[0010] The Chinese patent application CN 104 597 230 suggests a
method for manufacturing a functionalized polymer film, comprising
the following steps: forming a terminally functionalized
self-assembly mono-molecular layer on a surface of the substrate of
a biochip, e.g. a terminally functionalized thiol or dithiol
compound linked to a gold surface; grafting a photo-cross-linker to
the terminal of the self-assembly mono-molecular layer by chemical
bonding, e.g. a phenyldiazirine; spin-coating a polymer solution on
the resulting surface formed; and performing an UV irradiation on
the biochip having the spin-coated polymer surface to form a
chemical bonding under the UV light to have the polymer grafted to
the surface to form a polymer film.
SHORT SUMMARY OF THE INVENTION
[0011] Accordingly the invention relates to a method for the
immobilization of biomolecules containing at least one sulfhydryl
group, which method comprises the steps of: [0012] a) optionally
treating a biomolecule with an reducing agent in order to cleave
existing --S--S-- bridges in the biomolecule, or [0013] b)
optionally treating a biomolecule with an acylation agent carrying
a protected sulfhydryl group and deprotecting the sulfhydryl group;
[0014] c) contacting a modified metal surface with the biomolecule;
[0015] d) irradiating the resulting surface with UV radiation in
the presence of a photo-initiator, wherein said metal surface is
modified with a cross-linker compound comprising: [0016] i) a
terminal thiol or dithiol group covalently linked to the metal
surface being connected to [0017] ii) a spacer group, which at the
other terminal end is carrying an isolated C--C-double or
C--C-triple bond.
[0018] Furthermore, the invention relates to a compound of formula
(I),
HS--(CH.sub.2).sub.m--CH(ZH)-SPACER-(CH.sub.2).sub.p-A (I)
in which m is an integer from 2 to 6, A is selected from
--CH.dbd.CH.sub.2 and --C.ident.CH, and Z is S or a single bond,
SPACER is a group of formula
--(CH.sub.2).sub.n--(C.dbd.O).sub.x--Y--(CH.sub.2CH.sub.2--O).sub.y--(CH-
.sub.2).sub.r--(C.dbd.O).sub.v--X--
[0019] wherein
[0020] X and Y are each independently NH or 0,
[0021] n is 0 or an integer from 1 to 10,
[0022] x and v are each independently 0 or 1,
[0023] y is an integer from 1 to 20,
[0024] r and p are each independently selected from an integer from
1 to 6.
Another aspect of the invention is an intermediate of formula
(II),
##STR00001##
wherein A, SPACER, m and s have the meaning given for formula
(I).
[0025] Furthermore, the invention relates to a modified metal
surface in which at least one thiol group of the compound of
formula (I) according to the invention is covalently linked to at
least one of the metal atoms of the surface.
[0026] A final aspect of the invention is a kit for carrying out
the method of immobilizing biomolecules, in accordance with the
invention, said kit comprising [0027] i) a substrate with a
modified metal surface according to the invention, [0028] ii) an
optional containment unit containing a suitable reducing agent, or
[0029] iii) an optional containment unit containing a suitable
acylation agent carrying a protected sulfhydryl group and a
deprotection agent for the sulfhydryl group [0030] iv) a
containment unit containing a suitable photo-initiator, and [0031]
v) a leaflet explaining the conditions for carrying out the
method.
SHORT DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows fluorescence pictures of complementary
metal-oxide-semiconductor (CMOS) chips according to the invention
under different conditions in comparison with a surface modified
with lipoamide-PEG(11)-maleimide. A shows a
lipoamide-PEG(11)-maleimide modified surface with 10 min UV
irradiation at 304 nm wavelength. B shows an
R-.alpha.-lipoic-acid-PEG12-propargyl modified surface according to
the invention with 7.5 min irradiation time. C shows an
R-.alpha.-lipoic-acid-PEG12-propargyl modified surface without UV
irradiation. It is apparent that only little immobilization of the
antibody at the surface takes place without UV irradiation. D shows
Spotting-Layout KIA represents different reaction conditions of the
polyclonal rabbit anti-ACTH antibody. SPO represents the spotting
control, where only the spotting buffer has been applied.
[0033] FIG. 2 shows comparison of fluorescence pictures of CMOS
chips according to the invention on which a monoclonal mouse
antibody has been immobilized with and without use of a photo
initiator.
[0034] FIG. 3 shows fluorescence pictures of CMOS chips according
to the invention on which polyclonal rabbit anti-ACTH-antibodies
have been immobilized. A shows the immobilization of a polyclonal
rabbit anti-ACTH antibody with the photoreaction according to the
invention on different modified surfaces of R-.alpha.-lipoic-acid-5
kDa PEG-propargyl (Example 1.5) modified surface. B shows the
immobilization of a polyclonal rabbit anti-ACTH antibody with the
photoreaction according to the invention on different modified
surfaces of R-.alpha.-lipoic-acid-PEG12-propargyl (Example 1.2)
modified surface. C shows the immobilization of a polyclonal rabbit
anti-ACTH antibody with the photoreaction according to the
invention on different modified surfaces of R-.alpha.-lipoic-acid-5
kDa PEG-allyl (Example 1.4) modified surface. D shows the
immobilization of a polyclonal rabbit anti-ACTH antibody with the
photoreaction according to the invention on different modified
surfaces of R-.alpha.-lipoic-acid-PEG12-allyl (Example 1.1)
modified surface. E shows the spotting-layout KIA corresponds with
a polyclonal rabbit anti-ACTH antibody, wherein KIA shows the
highest concentration (100 .mu.g/mL as spotting solution), whereas
KIA4 contains the lowest concentration (12.5 .mu.g/mL as spotting
solution).
[0035] FIG. 4 shows a schematic representation of the
immobilization of a biomolecule according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] I. Listing of Terms
[0037] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN
0471186341); and other similar references.
[0038] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. Also, as used
herein, the term "comprises" means "includes". Hence "comprising A
or B" means including A, B, or A and B. It is further to be
understood that all nucleotide sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides or other compounds are approximate, and are
provided for description. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present disclosure, suitable methods and materials
are described below. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0039] To facilitate review of the various examples of this
disclosure, the following explanations of specific terms are
provided:
[0040] Biomolecule: A biologically active molecule, which may stem
from biological sources or may be produced synthetically.
[0041] Allergen: A nonparasitic antigen capable of stimulating a
type-I hypersensitivity reaction. Type I allergy is the production
of immunoglobulin E (IgE) antibodies against otherwise harmless
antigens, termed allergens, which can originate from a multitude of
allergen sources (e.g., mites, plant pollens, animals, insects,
molds, and food). IgE-mediated presentation of allergens to T cells
leads to T-cell activation and chronic allergic inflammation (e.g.,
chronic asthma, atopic dermatitis), particularly after repeated
contact with allergens. This event also induces increases of
allergen specific serum IgE levels and patients. Common allergens
include: those derived from plants, such as trees, for example
Betula verrucosa allergens Bet v I, Bet v 2, and Bet v 4;
Juniperous oxycedrus allergen Jun o 2; Castanea sativa allergen Cas
s 2; and Hevea brasiliensis allergens Hev b I, Hev b 3, Hev b 8,
Hev b 9, Hev b 10 and Hev b 11; grasses, such as Phleum pretense
allergens Phl p I, Phl p 2, Phl p 4, Phl p Sa, Phlp 5, Phlp 6, Phlp
7, Phl p 11, and Phl p 12; weeds, such as Parietaria Judaica
allergen Par j 2.01011; and Artemisia vulgaris allergens Art v I
and Art v 3; Mites, such as Dermatophagoides pteronyssinus
allergens Der p I, Der p 2, Der p 5, Der p 7, Der p 8, and Der p
10; Tyrophagu putrescentiae allergen Tyr p 2; Lepidoglyphus
destructor allergens Lep d 2.01 and Lep d 13; and Euroglyphus
maynei allergen Eur m 2.0101; animals, such as cats, for example
Felis domesticus allergen Fel d I; Penaeus aztecus allergen Pen a
I; Cyprinus carpo allergen Cyp c I; and albumin from cat, dog,
cattle, mouse, rat, pig, sheep, chicken, rabbit, hamster, horse,
pigeon, and guinea pig; Fungi, such as Penicillium citrinum
allergens Pen c 3 and Pen c 19; Penicillium notatum allergen
Penn13; Aspergillus fumigatus allergens Asp f I, Asp f3, Asp f4,
Asp f6, Asp f7 and Asp f8; Alternaria alternata allergens Alt a I
and Alt a 5; Malassezia furfur allergen Mal f I, Mal f 5, Mal f 6,
Mal f 7, Mal f 8, and Mal f 9; insects, such as Blatella germanica
allergens Bla g 2, Bla g 4, and Bla g 5; Apis mellifera allergens
Api m 2 and Api m I; Vespula vulgaris allergen Ves v 5; Vespula
germanica allergen Ves g 5; and Polstes annularis allergen Pol a 5;
food, such as Malus domestica allergens Mal d I and Mal d 2; Apium
graveolens allergens Api g I and Api g 1.0201; Daucus carota
allergen Dau c I; and Arachis hypogaea allergens Ara h 2 and Ara h
5 and the like. In some embodiments, an allergen or portion thereof
is part of a functionalized surface or electrode, thus a disclosed
functionalized surface can be used to measure the presence and
concentration of antibodies in a sample that specifically bind an
allergen. In some embodiments, an antibody that specifically binds
an allergen or portion thereof is part of a disclosed
functionalized surface or electrode, thus a disclosed
functionalized electrode can be used to measure the presence and
concentration of an allergen.
[0042] "Antibody" collectively refers to immunoglobulins or
immunoglobulin-like molecules (including by way of example and
without limitation, IgA, IgD, IgE, IgG and IgM, combinations
thereof), and similar molecules produced during an immune response
in any chordate such as a vertebrate, for example, in mammals such
as humans, goats, rabbits and mice and fragments thereof that
specifically bind to a molecule of interest (or a group of highly
similar molecules of interest) to the substantial exclusion of
binding to other molecules. An "antibody" typically comprises a
polypeptide ligand having at least a light chain or heavy chain
immunoglobulin variable region that specifically recognizes and
binds an epitope of an antigen. Exemplary antibodies include
polyclonal and monoclonal antibodies.
[0043] The term antibody also includes paratope sequences that are
able to bind other analytes.
[0044] Immunoglobulins are composed of a heavy and a light chain,
each of which has a variable region, termed the variable heavy (VH)
region and the variable light (VL) region. Together, the VH region
and the VL region are responsible for binding the antigen
recognized by the immunoglobulin. Exemplary immunoglobulin
fragments include, without limitation, proteolytic immunoglobulin
fragments (such as F(ab')2 fragments, Fab' fragments, Fab'-SH
fragments and Fab fragments as are known in the art), recombinant
immunoglobulin fragments (such as sFv fragments, dsFv fragments,
bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2
fragments), single chain Fv proteins ("scFv"), and disulfide
stabilized Fv proteins ("dsFv"). Other examples of antibodies
include diabodies, and triabodies (as are known in the art), and
camelid antibodies. "Antibody" also includes genetically engineered
molecules, such as chimeric antibodies. y. "Antibody" also includes
genetically engineered molecules, such as chimeric antibodies (for
example, humanized murine antibodies), and heteroconjugate
antibodies (such as, bispecific antibodies). See also, Pierce
Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,
Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New
York, 1997.
[0045] Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs." The extent of the framework region and CDRs
have been defined (see, Kabat et al., (1991) Sequences of Proteins
of Immunological Interest, 5th Edition, U.S. Department of Health
and Human Services, Public Health Service, National Institutes of
Health, Bethesda, Md. (NIH Publication No. 91-3242) which is hereby
incorporated by reference). The Kabat database is now maintained
online. The sequences of the framework regions of different light
or heavy chains are relatively conserved within a species. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three-dimensional space, for example
to hold the CDRs in an appropriate orientation for antigen
binding.
[0046] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDRI, CDR2 and CDR3, numbered sequentially starting from the N
terminus and are also typically identified by the chain in which
the particular CDR is located. Thus, a VH CDR3 is located in the
variable domain of the heavy chain of the antibody in which it is
found, whereas a VL CDRI is the CDRI from the variable domain of
the light chain of the antibody in which it is found.
[0047] A "monoclonal antibody" is an antibody produced by a single
clone of B-lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected or
transduced. Monoclonal antibodies are produced by methods known to
those of skill in the art, for instance by making hybrid
antibody-forming cells from a fusion of myeloma cells with immune
spleen cells. These fused cells and their progeny are termed
"hybridomas". Monoclonal antibodies include humanized monoclonal
antibodies. [0048] A "humanized" immunoglobulin, is an
immunoglobulin including a human framework region and one or more
CDRs from a non-human (such as a mouse, rat or synthetic)
immunoglobulin. The non-human immunoglobulin providing the CDRs is
termed a "donor", and the human immunoglobulin providing the
framework is termed an "acceptor". In one embodiment, all the CDRs
are from the donor immunoglobulin in a humanized immunoglobulin.
Constant regions need not be present, but if they are, they must be
substantially identical to human immunoglobulin constant regions,
for example at least about 85-90%, such as about 95% or more
identical. Hence, all parts of a humanized immunoglobulin, except
possibly the CDRs, are substantially identical to corresponding
parts of natural human immunoglobulin sequences. A "humanized
antibody" is an antibody comprising a humanized light chain and a
humanized heavy chain immunoglobulin. A humanized antibody binds to
the same antigen as the donor antibody that provides the CDRs. The
acceptor framework of a humanized immunoglobulin or antibody may
have a limited number of substitutions by amino acids taken from
the donor framework. Humanized or other monoclonal antibodies can
have additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin
functions. Humanized immunoglobulins can be constructed by means of
genetic engineering (for example see U.S. Pat. No. 5,585,089).
[0048] In some embodiments, an antibody specifically binds an
antigen of interest, such as an antigen that is part of a disclosed
functionalized electrode, for example covalently bonded to a thiol
or dithiol compound or a functionalized thiol or dithiol compound
that itself is bonded to an electrode surface. In some embodiments,
an antibody specific for an antigen of interest is part of a
disclosed functionalized electrode for example covalently bonded to
a thiol or dithiol compound or a functionalized thiol or dithiol
compound that itself is bonded to an electrode surface. In some
embodiments, an antibody is part of a detection reagent that
includes an enzyme.
[0049] Antigen: A compound, composition, or substance that may be
specifically bound by the products of specific humoral or cellular
immunity, such as an antibody molecule or T-cell receptor. Antigens
can be any type of molecule including, for example, haptens, simple
intermediary metabolites, sugars (e.g., oligosaccharides), lipids,
and hormones as well as macromolecules such as complex
carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids
and proteins. Common categories of antigens include, but are not
limited to, viral antigens, bacterial antigens, fungal antigens,
protozoa and other parasitic antigens, tumor antigens, antigens
involved in autoimmune disease, allergy and graft rejection,
toxins, and other antigens known in the art.
[0050] In some embodiments, an antigen is a ligand for an antibody
of interest, such as an antibody that is part of a disclosed
functionalized electrode, for example covalently bonded to a thiol
or dithiol compound or a functionalized thiol or dithiol compound
that itself is bonded to an electrode surface. In some embodiments,
an antigen of interest is part of a disclosed functionalized
electrode, for example covalently bonded to a thiol or dithiol
compound or a functionalized thiol or dithiol compound that itself
is bonded to an electrode surface.
[0051] Aptamer: Small nucleic acid and peptide molecules that bind
a specific target molecule, such as a target biomolecule, for
example an analyte, such as a target analyte. In some examples an
aptamer is part of a disclosed modified surface such as a
functionalized electrode.
[0052] Bacterial pathogen: A bacteria that causes disease
(pathogenic bacteria). Examples of pathogenic bacteria from which
antigens for use in the disclosed functionalized electrodes can be
derived include without limitation any one or more of (or any
combination of) Acinetobacter baumanii, Actinobacillus sp.,
Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and
Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas
hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and
Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes
xylosoxidans, Acinetobacter baumanii, Actinobacillus
actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis,
Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and
Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides
fragilis), Bartonella sp. (such as Bartonella bacilliformis and
Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as
Bordetella pertussis, Bordetella parapertussis, and Bordetella
bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and
Borrelia burgdorferi), Brucella sp. (such as Brucella abortus,
Brucella canis, Brucella melintensis and Brucella suis),
Burkholderia sp. (such as Burkholderia pseudomallei and
Burkholderia cepacia), Campylobacter sp. (such as Campylobacter
jejuni, Campylobacter coli, Campylobacter lari and Campylobacter
fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia
trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,
Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as,
Corynebacterium diphtheriae, Corynebacterium jeikeum and
Corynebacterium), Clostridium sp. (such as Clostridium perfringens,
Clostridium docile, Clostridium botulinum and Clostridium tetani),
Eikenella corrodens, Enterobacter sp. (such as Enterobacter
aerogenes, Enterobacter agglomerans, Enterobacter cloacae and
Escherichia coli, including opportunistic Escherichia coli, such as
enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic
E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and
uropathogenic E. coli) Enterococcus sp. (such as Enterococcus
faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia
chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae,
Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum,
Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such
as Haemophilus injluenzae, Haemophilus ducreyi, Haemophilus
aegyptius, Haemophilus parainjluenzae, Haemophilus haemolyticus and
Haemophilus parahaemolyticus), Helicobacter sp. (such as
Helicobacter pylori, Helicobacter cinaedi and Helicobacter
fennelliae), Eingella kingii, Elebsiella sp. (such as Elebsiella
pneumoniae, Elebsiella granulomatis and Elebsiella oxytoca),
Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans,
Legionella pneumophila, Leptospira interrogans, Peptostreptococcus
sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp.,
Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,
Mycobacterium tuberculosis, Mycobacterium intracellulare,
Mycobacterium avium, Mycobacterium bovis, and Mycobacterium
marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma
hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia
asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis),
Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria
meningitidis), Pasteurella multocida, Plesiomonas shigelloides.
Prevotella sp., Porphyromonas sp., Prevotella melami nogenica,
Proteus sp. (such as Proteus vulgaris and Proteus mirabilis),
Providencia sp. (such as Providencia alcalifaciens, Providencia
rettgeri and Providencia stuartii), Pseudomonas aeruginosa,
Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as
Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,
Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and
Rickettsia typhi), Rhodococcus sp., Serratia marcescens,
Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella
enterica, Salmonella typhi, Salmonella paratyphi, Salmonella
enteritidis, Salmonella cholerasuis and Salmonella typhimurium),
Serratia sp. (such as Serratia marescens and Serratia
liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella
flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp.
(such as Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus hemolyticus, Staphylococcus saprophyticus),
Streptococcus sp. (such as Streptococcus pneumoniae (for example
chloramphenicol resistant serotype 4 Streptococcus pneumoniae,
spectinomycin-resistant serotype 6B Streptococcus pneumoniae,
streptomycin-resistant serotype 9V Streptococcus pneumoniae,
erythromycin-resistant serotype 14 Streptococcus pneumoniae,
optochin-resistant serotype 14 Streptococcus pneumoniae,
rifampicin-resistant serotype 18C Streptococcus pneumoniae,
tetracycline-resistant serotype 19F Streptococcus pneumoniae,
penicillin-resistant serotype 19F Streptococcus pneumoniae, and
trimethoprim-resistant serotype 23F Streptococcus pneumoniae,
chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,
spectinomycin-resistant serotype 6B Streptococcus pneumoniae,
streptomycin-resistant serotype 9V Streptococcus pneumoniae,
optochin-resistant serotype 14 Streptococcus pneumoniae,
rifampicin-resistant serotype 18C Streptococcus pneumoniae,
penicillin-resistant serotype 19F Streptococcus pneumoniae, or
trimethoprim resistant serotype 23F Streptococcus pneumoniae),
Streptococcus agalactiae, Streptococcus mutans, Streptococcus
pyogenes, Group A streptococci, Streptococcus pyogenes, Group B
streptococci, Streptococcus agalactiae, Group C streptococci,
Streptococcus anginosus, Streptococcus equismilis, Group D
streptococci, Streptococcus bovis, Group F streptococci, and
Streptococcus anginosus Group G streptococci), Spirillum minus,
Streptobacillus moniliformi, Treponema sp. (such as Treponema
carateum, Treponema petenue, Treponema pallidum and Treponema
endemicum, Tropheryma whippelii, Ureaplasma urealyticum,
Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio
parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio
vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae,
Vibrio fiuvialis, Vibrio metchnilrovii, Vibrio damsela and Vibrio
furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia
pestis, and Yersinia pseudotuberculosis) and Xanthomonas
maltophilia among others.
[0053] Bacterial antigens suitable for use in the disclosed methods
and compositions include proteins, polysaccharides,
lipopolysaccharides, and outer membrane vesicles which may be
isolated, purified or derived from a bacterium. In addition,
bacterial antigens include bacterial lysates and inactivated
bacteria formulations. Bacteria antigens can be produced by
recombinant expression. Bacterial antigens preferably include
epitopes which are exposed on the surface of the bacteria during at
least one stage of its life cycle. Bacterial antigens include but
are not limited to antigens derived from one or more of the
bacteria set forth above as well as the specific antigens examples
identified below.
[0054] Neiserria gonorrhoeae antigens include Por (or porn)
protein, such as PorB (see, e.g., Zhu et al. (2004) Vaccine
22:660-669), a transferring binding protein, such as TbpA and TbpB
(see, e.g., Price et al. (2004) Infect. Immun. 71(1):277-283), an
opacity protein (such as Opa), a reduction-modifiable protein
(Rmp), and outer membrane vesicle (OMV) preparations (see, e.g.,
Plante et al. (2000) J. Infect. Dis. 182:848-855); WO 99/24578; WO
99/36544; WO 99/57280; and WO 02/079243, all of which are
incorporated by reference).
[0055] Chlamydia trachomatis antigens include antigens derived from
serotypes A, B, Ba and C (agents of trachoma, a cause of
blindness), serotypes Li, L3 (associated with Lymphogranuloma
venereum), and serotypes, D-K. Chlamydia trachomas antigens also
include antigens identified in WO 00/37494; WO 03/049762; WO
03/068811; and WO 05/002619 (all of which are incorporated by
reference), including PepA (CT045), LcrE (CT089), Art (CT381), DnaK
(CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA (CT444),
AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), MurG (CT761),
CT396 and CT761, and specific combinations of these antigens.
[0056] Treponema pallidum (Syphilis) antigens include TmpA
antigen.
[0057] The compositions of the disclosure can include one or more
antigens derived from a sexually transmitted disease (STD). Such
antigens can provide for prophylactis or therapy for STDs such as
chlamydia, genital herpes, hepatitis (such as HCV), genital warts,
gonorrhea, syphilis and/or chancroid (see WO 00/15255, which is
incorporated by reference). Antigens may be derived from one or
more viral or bacterial STDs. Viral STD antigens for use in the
invention may be derived from, for example, HIV, herpes simplex
virus (HSV-I and HSV-2), human papillomavirus (HPV), and hepatitis
(HCV). Bacterial STD antigens for use in the invention may be
derived from, for example, Neiserria gonorrhoeae, Chlamydia
trachomatis, Treponema pallidum, Haemophilus ducreyi, E. coli, and
Streptococcus agalactiae.
[0058] In some embodiments, a disclosed functionalized surface or
electrode includes one or more antigens derived from one or more of
the organisms listed above. In some embodiments, an antibody that
specifically binds antigens derived from one or more of the
organisms listed above is part of a disclosed functionalized
electrode, and thus in some examples can be used to detect such
antigens in a sample, for example to diagnose a particular
bacterial infection.
[0059] Binding affinity: Affinity of a specific binding agent for
its target, such as an antibody for an antigen, for example an
antibody for a target analyte, such as a target analyte. In one
embodiment, affinity is calculated by a modification of the
Scatchard method described by Frankel et al., Mol. Immunol.,
16:101-106, 1979. In another embodiment, binding affinity is
measured by a specific binding agent receptor dissociation rate. In
yet another embodiment, a high binding affinity is measured by a
competition radioimmunoassay. In several examples, a high binding
affinity (K.sub.D) is at least about 1.times.10.sup.-8 M. In other
embodiments, a high binding affinity is at least about
1.5.times.10.sup.-8, at least about 2.0.times.10.sup.-8, at least
about 2.5.times.10.sup.-8, at least about 3.0.times.10.sup.-8, at
least about 3.5.times.10.sup.-8, at least about
4.0.times.10.sup.-8, at least about 4.5.times.10.sup.-8 or at least
about 5.0.times.10.sup.-8 M.
[0060] Biomolecule: Any molecule that was derived from biological
system, including but not limited to, a synthetic or naturally
occurring protein, glycoprotein, lipoprotein, amino acid,
nucleoside, nucleotide, nucleic acid, oligonucleotide, DNA, PNA,
RNA, carbohydrate, sugar, lipid, fatty acid, hapten, antibiotics,
vitamins, enterotoxins and the like. In some examples, a
biomolecule is a target analyte for which the presence and or
concentration or amount can be determined. In some embodiments a
biomolecule is covalently bonded to a thiol or dithiol compound,
and/or a cross-linker, such as a thiol or dithiol compound that is
part of a disclosed functionalized metal surface, in particular an
electrode.
[0061] Chemokines: Proteins classified according to shared
structural characteristics such as small size (approximately 8-10
kilodaltons (kDa) in mass) and the presence of four cysteine
residues in conserved locations that are key to forming their
3-dimensional shape. These proteins exert their biological effects
by interacting with G protein-linked trans-membrane receptors
called chemokine receptors that are selectively found at the
surfaces of their target cells. Chemokines bind to chemokine
receptors and thus are chemokine receptor ligands.
[0062] Examples of chemokines include the CCL chemokines such as
CCL1, CCL2, CCL3, CCL4, CCLS, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11,
CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,
CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27 and CCL28; CXCL
chemokines such as CXCL1, CXCL2, CXCL3, CXCL4, CXCLS, CXCL6, CXCL7,
CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,
CXCL16 and CXCL17; XCL chemokines such as XCL1 and XCL2; and CX3CL
chemokines such as CX3CL1. In some embodiments, a chemokine or
portion thereof is part of a disclosed functionalized electrode. In
some embodiments, an antibody that specifically binds a chemokine
or portion thereof is part of a functionalized electrode, and thus
in some examples can be used to detect such chemokines in a
sample.
[0063] Conjugating, joining, bonding or linking: Chemically
coupling a first unit to a second unit. This includes, but is not
limited to, covalently bonding one molecule to another molecule,
non-covalently bonding one molecule to another (e.g.,
electro-statically bonding), non-covalently bonding one molecule to
another molecule by hydrogen bonding, non-covalently bonding one
molecule to another molecule by van der Waals forces, and any and
all combinations of such couplings. In some embodiments a ligand
for a target analyte is covalently bonded to a thiol or dithiol
compound, and/or a cross-linker.
[0064] Contacting: Placement in direct physical association
including both in solid or liquid form.
[0065] Control: A reference standard. In some examples, a control
can be a known value indicative of a known concentration or amount
of an analyte, such as a target analyte for example a biomolecule
of interest. In some examples a control, or a set of controls of
known concentration or amount, can be used to calibrate a
functionalized electrode.
[0066] A difference between a test sample and a control can be an
increase or conversely a decrease. The difference can be a
qualitative difference or a quantitative difference, for example a
statistically significant difference. In some examples, a
difference is an increase or decrease, relative to a control, of at
least about 10%, such as at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
100%, at least about 150%, at least about 200%, at least about
250%, at least about 300%, at least about 350%, at least about
400%, at least about 500%, or greater than 500%.
[0067] Complex (complexed): Two proteins, or fragments or
derivatives thereof, one protein (or fragment or derivative) and a
non-protein compound, molecule or any two or more compounds are
said to form a complex when they measurably associate with each
other in a specific manner. In some examples, a complex is the
complex formed between a functionalized electrode and a target
analyte.
[0068] Covalent bond: An interatomic bond between two atoms,
characterized by the sharing of one or more pairs of electrons by
the atoms. The terms "covalently bound" or "covalently linked"
refer to making two separate molecules into one contiguous
molecule, for example ligand specific for a target analyte and a
thiol or dithiol compound can be covalently linked (such as
directly or indirectly through a linker).
[0069] Cross-linker: A homo- or hetero-multifunctional reagent with
at least two non-identical groups, which are reactive to at least
one functional group present in biomolecules, such as sulfhydryl
groups, in a photoreaction and another functional group which forms
a covalent bond to the metallic surface. Both functional groups are
as a rule separated from each other by a Spacer group. In some
examples, a protein cross-linker is sulfhydryl reactive, meaning it
is capable of forming a covalent bond with a sulfhydryl group, such
as an sulfhydryl group present in a biomolecule, for example a
sulfhydryl group present on a cysteine residue, or for example a
sulfhydryl group introduced by reacting an amine group with an
agent which carries a protected sulfhydryl group followed by
deprotection.
[0070] Cytokine: A generic name for a diverse group of soluble
proteins and peptides that act as humoral regulators at nano- to
pico-molar concentrations and which, either under normal or
pathological conditions, modulate the functional activities of
individual cells and tissues. These proteins also mediate
interactions between cells directly and regulate processes taking
place in the extracellular environment. Cytokines include both
naturally occurring peptides and variants that retain full or
partial biological activity. Cytokines bind to cytokine receptors
and thus are cytokine receptor ligands.
[0071] Examples of cytokines include interleukins, such as IL-1 a,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10 and IL-12; interferons,
such as IFN-.alpha., IFN-(3 and IFN-.gamma.; tumor necrosis
factors, such as TNF-.alpha. and TNF-(3 macrophage; inflammatory
proteins, such as MIP-1 a and MIP-1 3; and transforming growth
factors, such as TGF-(3. In some embodiments, a cytokine or portion
thereof is part of a disclosed functionalized electrode. In some
embodiments, an antibody that specifically binds a cytokine or
portion thereof is part of a disclosed functionalized electrode,
thus the presence of a cytokine in a sample can be determined using
a disclosed functionalized electrode.
[0072] Cyclic voltammetry: An electrochemical technique that can be
used to obtain information about the redox potential of analyte
solutions or enzyme substrate pairs, for example to select an
enzyme substrate pair for inclusion in a disclosed biosensor. The
voltage is swept between two values at a fixed rate, however, when
the voltage reaches V2 the scan is reversed and the voltage is
swept back to V1. The voltage is measured between a reference
electrode and the working electrode, while the current is measured
between the working electrode and the counter electrode. The
obtained measurements are plotted as current vs. voltage, also
known as a voltammogram. As the voltage is increased toward the
electrochemical reduction potential of the analyte, the current
will also increase. With increasing voltage toward V2 past this
reduction potential, the current decreases, having formed a peak,
since the oxidation potential has been exceeded. As the voltage is
reversed to complete the scan toward V1, the reaction will begin to
reoxidize the product from the initial reaction. This produces an
increase in current of opposite polarity as compared to the forward
scan, but again decreases having formed a second peak as the
voltage scan continues toward V1. The reverse scan also provides
information about the reversibility of a reaction at a given scan
rate. The shape of the voltammogram for a given compound depends
not only on the scan rate and the electrode surface, which is
different after each adsorption step, but can also depend on the
catalyst concentration.
[0073] Detect: To determine if an agent (such as a signal or target
analyte) is present or absent. In some examples, this can further
include quantification. In some examples, an electro-magnetic
signal is used to detect the presence, amount or concentration of
an agent, such as an analyte. In some examples, the detection is
indirect, for example using an enzyme that catalyzes the production
of a detectable signal when an analyte is present. In other
examples, the signal is reduced when the analyte is present, such
that increasing concentration of an analyte gives a decrease in
signal.
[0074] Dithiol group: A terminal group of a cross-linker which
exhibits two thiol or sulfhydryl groups as a rule separated by a
C.sub.1-5 alkylenediyl group. Most preferred are those dithiols,
which can be obtained by reduction of a liponic acid
derivative.
[0075] Epitope: An antigenic determinant. These are particular
chemical groups or contiguous or non-contiguous peptide sequences
on a molecule that are antigenic, that is, that elicit a specific
immune response. An antibody binds a particular antigenic epitope
based on the three dimensional structure of the antibody and the
matching (or cognate) epitope.
[0076] Electromagnetic radiation: A series of electromagnetic waves
that are propagated by simultaneous periodic variations of electric
and magnetic field intensity, and that includes radio waves,
infrared, visible light, ultraviolet (UV) light, X-rays and gamma
rays. In particular examples, electromagnetic is in the form of
electrons, which can be detected as a change in current in an
electrode, for example the functionalized metal surfaces disclosed
herein.
[0077] Fungal pathogen: A fungus that causes disease. Examples of
fungal pathogens for use in accordance with the disclosed methods
and compositions include without limitation any one or more of (or
any combination of) Trichophyton rubrum, T. mentagrophytes,
Epidermophyton floccosum, Microsporum canis, Pityrosporum
orbiculare (Malassezia furfur), Candida sp. (such as Candida
albicans), Aspergillus sp. (such as Aspergillus fumigatus,
Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp.
(such as Cryptococcus neoformans, Ciyptococcus gattii, Ciyptococcus
laurentii and Ciyptococcus albidus), Histoplasma sp. (such as
Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis
jirovecii), and Stachybotrys (such as Stachybotrys chartarum). In
some embodiments, a disclosed functionalized substrate or electrode
includes one or more antigens derived from one or more of the
organisms listed above. In some embodiments, an antibody that
specifically binds antigens derived from one or more of the
organisms listed above is part of a disclosed functionalized
electrode, and thus in some examples can be used to detect such
antigens in a sample, for example to diagnose a particular fungal
infection or the presence of a fungus in an environmental
sample.
[0078] Growth factor: Proteins capable of stimulating cellular
proliferation and cellular differentiation. Examples of growth
factors include transforming growth factor beta (TGF-(3),
granulocyte-colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), nerve
growth factor (NGF), neurotrophins, platelet-derived growth factor
(PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin
(GDF-8), growth differentiation factor-9 (GDF-9), basic fibroblast
growth factor (bFGF or FGF2), epidermal growth factor (EGF),
hepatocyte growth factor (HGF) and the like. In some embodiments, a
growth factor or portion thereof is part of a disclosed
functionalized electrode. In some embodiments, an antibody that
specifically binds a growth factor or portion thereof is part of a
disclosed functionalized electrode and thus in some examples can be
used to detect such growth factors in a sample.
[0079] Heterologous: With reference to a molecule, such as a
linker, "heterologous" refers to molecules that are not normally
associated with each other, for example as a single molecule. Thus,
a "heterologous" linker is a linker attached to another molecule
that the linker is usually not found in association with in nature,
such as in a wild-type molecule.
[0080] High throughput technique: Through this process, one can
rapidly identify analytes present in a sample or multiple samples.
In certain examples, combining modern robotics, data processing and
control software, liquid handling devices, and sensitive detectors,
high throughput techniques allows the rapid detection and/or
quantification of an analyte in a short period of time, for example
using the assays and compositions disclosed herein.
[0081] Hormone: A classification of small molecules that carries a
signal from one cell (or group of cells) to another. Examples of
hormones include amine-tryptophans, such as melatonin
(n-acetyl-5-methoxytryptamine) and serotonin; amine-tyrosines, such
as thyroxine (thyroid hormone), tri-iodothyronine (thyroid
hormone), epinephrine (adrenaline), norepinephrine (noradrenaline)
and dopamine; peptide hormones, such as antimullerian hormone
(mullerian inhibiting factor), adiponectin, adrenocorticotropic
hormone (orticotropin), angiotensinogen and angiotensin,
antidiuretic hormone (vasopressin, arginine vasopressin),
atrial-natriuretic peptide atriopeptin), calcitonin,
cholecystokinin, corticotropin-releasing hormone, erythropoietin,
follicle-stimulating hormone, gastrin, ghrelin, glucagon,
gonadotropin-releasing hormone, growth hormone-releasing hormone,
human chorionic gonadotropin, human placental lactogen, growth
hormone, inhibin, insulin, insulin-like growth factor
(somatomedin), leptin, luteinizing hormone, melanocyte stimulating
hormone, oxytocin, parathyroid hormone, prolactin, relaxin,
secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone
and thyrotropin-releasing hormone; steroids, such as cortisol,
aldosterone, testosterone, dehydroepiandrosterone, androstenedione,
dihydrotestosterone, estradiol, estrone, estriol, progesterone and
calcitriol (Vitamin d3); and eicosanoids, such as prostaglandins,
leukotrienes, prostacyclin and thromboxane, among others. In some
embodiments, a hormone or portion thereof is part of a disclosed
functionalized electrode. In some embodiments, an antibody that
specifically binds a hormone or portion thereof is part of
disclosed functionalized electrode. Thus in some examples the
disclosed functionalized electrodes can be used to detect such
hormones and the pre-cursors and analogues thereof.
[0082] Isolated: An "isolated" biological component (such as a
biomolecule) has been substantially separated or purified away from
other components in a mixture.
[0083] Ligand: Any molecule which specifically binds an analyte of
interest (for example a target analyte), such as an antibody,
protein, peptide or a small molecule (for example a molecule with a
molecular mass less than 10 kilodaltons, (kDa) that specifically
binds an analyte, such as a target analyte).
[0084] Linker or cross-linker: A compound or moiety that acts as a
molecular bridge to operably link two different molecules, wherein
one portion of the linker is operably linked to a first molecule
and wherein another portion of the linker is operably linked to a
second molecule. The two different molecules can be linked to the
linker in a stepwise manner. There is no particular size or content
limitations for the linker so long as it can fulfil its purpose as
a molecular bridge. Linkers are known to those skilled in the art
to include, but are not limited to, chemical chains, chemical
compounds, carbohydrate chains, peptides, haptens and the like. The
linkers can include, but are not limited to, homobifunctional
linkers and heterobifunctional linkers. Heterobifunctional linkers,
well known to those skilled in the art, contain one end having a
first reactive functionality to specifically link a first molecule
and an opposite end having a second reactive functionality to
specifically link to a second molecule. Depending on such factors
as the molecules to be linked and the conditions in which the
method of detection is performed, the linker can vary in length and
composition for optimizing such properties as flexibility,
stability and resistance to certain chemical and/or temperature
parameters.
[0085] Nucleic acid: A polymer composed of nucleotide units
(ribonucleotides, deoxyribonucleotides, related naturally occurring
structural variants and synthetic non-naturally occurring analogues
thereof or combinations thereof) linked via phosphodiester bonds,
related naturally occurring structural variants and synthetic
non-naturally occurring analogues thereof. Thus, the term includes
nucleotide polymers in which the nucleotides and the linkages
between them include non-naturally occurring synthetic analogs,
such as, for example and without limitation, phosphorothiolates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) and the
like. Such polynucleotides can be synthesized, for example, using
an automated DNA synthesizer. The term "oligonucleotide" typically
refers to short polynucleotides, generally no greater than about 50
nucleotides. It will be understood that when a nucleotide sequence
is represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces
"T."
[0086] Conventional notation is used herein to describe nucleotide
sequences: the left-hand end of a single-stranded nucleotide
sequence is the 5'-end; the left-hand direction of a
double-stranded nucleotide sequence is referred to as the
5'-direction. The direction of 5' to 3' addition of nucleotides to
nascent RNA transcripts is referred to as the transcription
direction. The DNA strand having the same sequence as an mRNA is
referred to as the "coding strand;" sequences on the DNA strand
having the same sequence as an mRNA transcribed from that DNA and
which are located 5' to the 5'-end of the RNA transcript are
referred to as "upstream sequences;" sequences on the DNA strand
having the same sequence as the RNA and which are 3' to the 3' end
of the coding RNA transcript are referred to as "downstream
sequences."
[0087] "Recombinant nucleic acid" refers to a nucleic acid having
nucleotide sequences that are not naturally joined together. This
includes nucleic acid vectors comprising an amplified or assembled
nucleic acid which can be used to transform a suitable host cell. A
host cell that comprises the recombinant nucleic acid is referred
to as a "recombinant host cell." The gene is then expressed in the
recombinant host cell to produce, for example a "recombinant
polypeptide." A recombinant nucleic acid may serve a non-coding
function (for example a promoter, origin of replication,
ribosome-binding site, etc.) as well.
[0088] For sequence comparison of nucleic acid sequences, typically
one sequence acts as a reference sequence, to which test sequences
are compared. When using a sequence comparison algorithm, test and
reference sequences are entered into a computer, subsequence
coordinates are designated, if necessary and sequence algorithm
program parameters are designated. Default program parameters are
used. Methods of alignment of sequences for comparison are well
known in the art. Optimal alignment of sequences for comparison can
be conducted, for example, by the local homology algorithm of Smith
& Waterman, Adv. Appl. Math. 2:482, 1981, by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
48:443, 1970, by the search for similarity method of Pearson &
Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr., Madison, Wis.) or by manual alignment and
visual inspection (see, for example, Current Protocols in Molecular
Biology (Ausubel et al., eds 1995 supplement)).
[0089] Nucleotide: The fundamental unit of nucleic acid molecules.
A nucleotide includes a nitrogen-containing base attached to a
pentose monosaccharide with one, two or three phosphate groups
attached by ester linkages to the saccharide moiety.
[0090] The major nucleotides of DNA are deoxyadenosine
5'-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP
or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine
5'-triphosphate (dTTP or T). The major nucleotides of RNA are
adenosine 5'-triphosphate (ATP or A), guanosine 5'-triphosphate
(GTP or G), cytidine 5'-triphosphate (CTP or C) and uridine
5'-triphosphate (UTP or U).
[0091] Nucleotides include those nucleotides containing modified
bases, modified sugar moieties and modified phosphate backbones,
for example as described in U.S. Pat. No. 5,866,336.
[0092] Examples of modified base moieties which can be used to
modify nucleotides at any position on its structure include, but
are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, acetyl-cytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N-6-sopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-meth-ylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxy-acetic acid methylester, uracil-5-oxyacetic acid,
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and
2,6-diaminopurine 2'-deoxyguanosine amongst others.
[0093] Examples of modified sugar moieties, which may be used to
modify nucleotides at any position on its structure, include, but
are not limited to arabinose, 2-fluoroarabinose, xylose and hexose
or a modified component of the phosphate backbone, such as
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate or an
alkyl phosphotriester or analog thereof.
[0094] Neuropeptide: Peptides released by neurons in the mammalian
brain that specifically bind a neuropeptide receptor. Examples of
neuropeptides include a-melanocyte-stimulating hormone (a-MSH),
galanin-like peptide, a cocaine- and-amphetamine-regulated
transcript (CART), neuropeptide Y, agouti-related peptide (AGRP),
(3-endorphin, dynorphin, enkephalin, galanin, ghrelin,
growth-hormone releasing hormone, neurotensin, neuromedin U,
somatostatin, galanin, enkephalin cholecystokinin, vasoactive
intestinal polypeptide (VIP) and substance P among others. In some
embodiments, a neuropeptide or portion thereof is part of a
disclosed functionalized electrode. In some embodiments, an
antibody that specifically binds a neuropeptide or portion thereof
is part of a functionalize electrode, and thus in some examples can
be used to detect such peptides in a sample.
[0095] Oligonucleotide: A linear polynucleotide sequence of up to
about 100 nucleotide bases in length.
[0096] Parasite: An organism that lives inside humans or other
organisms acting as hosts (for the parasite). Parasites are
dependent on their hosts for at least part of their life cycle.
Parasites are harmful to humans because they consume needed food,
eat away body tissues and cells, and eliminate toxic waste, which
makes people sick. Examples of parasites for use in accordance with
the disclosed methods and compositions include without limitation
any one or more of (or any combination of) Malaria (Plasmodium
falciparum, P vivax, P malariae), Schistosomes, Trypanosomes,
Leishmania, Filarial nematodes, Trichomoniasis, S arcosporidiasis,
Taenia (T. saginata, T. solium), Toxoplasma gondii, Trichinelosis
(Trichinella spiralis) or Coccidiosis (Eimedia species). Thus in
some embodiments, a disclosed functionalized electrode includes one
or more antigens derived from one or more of the organisms listed
above. In some embodiments, an antibody that specifically binds
antigens derived from one or more of the organisms listed above is
part of a disclosed functionalized electrode. Thus in some examples
a disclosed functionalized electrode can be used to detect such
parasites in a sample, for example to diagnose a particular
parasitic infection or the presence of parasites in an
environmental sample.
[0097] Photo-initiator: An organic molecule or group, which is
cleaved into separate radical groups upon irradiation with UV
light. Preferred are such molecules or groups which derive from
.alpha.-hydroxy-, .alpha.-alkoxy- or .alpha.-amino-arylketons,
preferably they exhibit a 1-benzoyl-1-methyl-ethanol moiety; most
preferred photo-initiators are
2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and
2,2-dimethoxy-2-phenylphenone. In "the presence of a
photo-initiator" means that a photo-initiator is either added in
the form of a solution prior to the photoreaction or has been
previously attached to the metal surface as disclosed for example
by U.S. Pat. No. 8,580,571.
[0098] Polypeptide: A polymer in which the monomers are amino acid
residues, which are joined together through amide bonds. When the
amino acids are a-amino acids, either the L-optical isomer or the
D-optical isomer can be used. The terms "polypeptide" or "protein"
as used herein are intended to encompass any amino acid sequence
and include modified sequences such as glycoproteins. "Polypeptide"
covers naturally occurring proteins, as well as those which are
recombinantly or synthetically produced. "Residue" or "amino acid
residue" includes an amino acid that is incorporated into a
protein, polypeptide, or peptide.
[0099] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified peptide, protein, conjugate, or other compound
is one that is isolated in whole or in part from proteins or other
constituents of a mixture. Generally, substantially purified
peptides, proteins, conjugates, or other active compounds for use
within the disclosure comprise more than 80% of all macromolecular
species present in a preparation prior to admixture or formulation
of the peptide, protein, conjugate or other active compound with a
pharmaceutical carrier, excipient, buffer, absorption enhancing
agent, stabilizer, preservative, adjuvant or other co-ingredient.
More typically, the peptide, protein, conjugate or other active
compound is purified to represent greater than 90%, often greater
than 95% of all macromolecular species present in a purified
preparation prior to admixture with other formulation ingredients.
In other cases, the purified preparation may be essentially
homogeneous, wherein other macromolecular species are not
detectable by conventional techniques.
[0100] Quantitating: Determining or measuring a quantity (such as a
relative quantity) of a molecule or the activity of a molecule,
such as the quantity of analyte, such as a target analyte present
in a sample.
[0101] Sample: A material to be analysed. In one embodiment, a
sample is a biological sample. In another embodiment, a sample is
an environmental sample, such as soil, sediment water, or air.
Environmental samples can be obtained from an industrial source,
such as a farm, waste stream, or water source. A biological sample
is one that includes biological materials (such as nucleic acid and
proteins). In some examples, a biological sample is obtained from
an organism or a part thereof, such as an animal. In particular
embodiments, the biological sample is obtained from an animal
subject, such as a human subject. A biological sample can be any
solid or fluid sample obtained from, excreted by or secreted by any
living organism, including without limitation multicellular
organisms (such as animals, including samples from a healthy or
apparently healthy human subject or a human patient affected by a
condition or disease to be diagnosed or investigated, such as
cancer). For example, a biological sample can be a biological fluid
obtained from, for example, blood, plasma, serum, urine, bile,
ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or
any bodily secretion, a transudate, an exudate (for example, fluid
obtained from an abscess or any other site of infection or
inflammation), or fluid obtained from a joint (for example, a
normal joint or a joint affected by disease, such as a rheumatoid
arthritis, osteoarthritis, gout or septic arthritis). A biological
sample can also be a sample obtained from any organ or tissue
(including a biopsy or autopsy specimen, such as a tumor biopsy) or
can include a cell (whether a primary cell or cultured cell) or
medium conditioned by any cell, tissue or organ. In some examples,
a biological sample is a cell lysate, for example a cell lysate
obtained from a tumor of a subject.
[0102] Spacer group: A group within the cross-linker, which
separates the two terminal reactive groups, one of which binds to
the metal surface and the other to a sulfhydryl group of the
biomolecule upon irradiation. The Spacer group is preferably a
C.sub.5-30 alkylene-di-1,.omega.-yl group, wherein one or more
non-adjacent CH.sub.2 groups may be replaced each independently by
a group selected from O, S, NH, NR, NR--CO, CO--NR, O--CO and
CO--O, wherein R represents hydrogen or C.sub.1-6 alkyl.
Polyethyleneglycol (PEG) groups are preferred components of such
Spacer groups.
[0103] Specific binding agent: An agent that binds substantially
only to a defined target. Thus, an antigen binding agent, such as
an antibody that is specific for an antigen is an agent that binds
substantially to a specific antigen or fragment thereof. In some
examples, the specific binding agent is a monoclonal or polyclonal
antibody that specifically binds a specific antigen or antigenic
fragment thereof, such as a target analyte. In other examples, the
specific binding agent is an antigen that specifically binds to an
antibody specific for the antigen. In some examples, a specific
binding agent is conjugated to an enzyme, such as an enzyme that
catalyzes the reaction of an enzyme Substrate into an electroactive
product.
[0104] Subject: Includes both human and veterinary subjects, for
example, humans, non-human primates, dogs, cats, horses, swine, and
cows, and further subjects of the field of food production such as
chicken or fishes such as salmon, tuna or trout. Other important
subjects in the field of food production are plant materials.
[0105] Substrate: A molecule that is acted upon by an enzyme. A
substrate binds with the enzyme's active site, and an
enzyme-substrate complex is formed. In some examples an enzyme
substrate is converted to an electroactive product by an
enzyme.
[0106] Thiol: An organosulfur compound that contains a
sulfur-hydrogen bond or sulfhydryl group (S--H). Thiols are the
sulfur analogue of an alcohol. The S--H functional group can be
referred to as either a thiol group or a sulfhydryl group. Thiols
have the general chemical formula R--S--H. In some examples, the
S--H group can react with and thereby bond to a surface, such as an
electrically conductive surface.
[0107] Tumor antigen: A tumor antigen is an antigen produced by
tumor cells that can stimulate tumor-specific T-cell immune
responses. Exemplary tumor antigens include, but are not limited
to, RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1,
glycoprotein (gp) 75, gp100, beta-catenin, preferentially expressed
antigen of melanoma (PRAME), MUM-1, Wilms tumor (WT)-1,
carcinoembryonic antigen (CEA), and PR-1. Additional tumor antigens
are known in the art (for example see Novellino et al., Cancer
Immunol. Immunother. 54(3):187-207, 2005) and are described below.
Tumor antigens are also referred to as "cancer antigens." The tumor
antigen can be any tumor-associated antigen, which are well known
in the art and include, for example, carcinoembryonic antigen
(CEA), (3-human chorionic gonadotropin, alphafetoprotein (AFP),
lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human
telomerase reverse transcriptase, RU1, RU2 (AS), intestinal
carboxyl esterase, mut hsp70-2, macrophage colony stimulating
factor, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1,
LAGE-la, p53, prostein, PSMA, Her2/neu, survivin and telomerase,
prostate-carcinoma tumor antigen-1, MAGE, ELF2M, neutrophil
elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II,
IGF-I receptor and mesothelin.
[0108] In some embodiments, a tumor antigen or portion thereof is
part of a disclosed functionalized electrode. In some embodiments,
an antibody that specifically binds a tumor antigen or portion
thereof is part of a functionalized electrode. Thus in some
examples the disclosed functionalized electrodes can be used to
detect such antigens in a sample, for example to diagnose a
cancer.
[0109] Virus: A microscopic infectious organism that reproduces
inside living cells. A virus consists essentially of a core of
nucleic acid surrounded by a protein coat, and has the ability to
replicate only inside a living cell. "Viral replication" is the
production of additional virus by the occurrence of at least one
viral life cycle. A virus may subvert the host cells' normal
functions, causing the cell to behave in a manner determined by the
virus. For example, a viral infection may result in a cell
producing a cytokine, or responding to a cytokine, when the
uninfected cell does not normally do so. In some examples, a virus
is a pathogen. Another form of viruses are prions.
[0110] Specific examples of viral pathogens for use in accordance
with the disclosed methods and compositions include without
limitation any one or more of (or any combination of); Arenaviruses
(such as Guanarito virus, Lassa virus, Junin virus, Machupo virus
and Sabia), Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses
(such as Crimean-Congo hemorrhagic fever virus and Hantavirus),
Barnaviruses, Birnaviruses, Bornaviruses (such as Borna disease
virus), Bromoviruses, Caliciviruses, Chrysoviruses, Coronaviruses
(such as Coronavirus and SARS), Cystoviruses, Closteroviruses,
Comoviruses, Dicistroviruses, Flaviruses (such as Yellow fever
virus, West Nile virus, Hepatitis C virus, and Dengue fever virus),
Filoviruses (such as Ebola virus and Marburg virus), Flexiviruses,
Hepeviruses (such as Hepatitis E virus), human adenoviruses (such
as human adenovirus A-F), human astroviruses, human BK
polyomaviruses, human bocaviruses, human coronavirus (such as a
human coronavirus HKU1, NL63, and 0C43), human enteroviruses (such
as human enterovirus A-D), human erythrovirus V9, human foamy
viruses, human herpesviruses (such as human herpesvirus 1 (herpes
simplex virus type 1), human herpes-virus 2 (herpes simplex virus
type 2), human herpesvirus 3 (Varicella zoster virus), human
herpesvirus 4 type 1 (Epstein-Barr virus type 1), human herpesvirus
4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5 strain
AD169, human herpesvirus 5 strain Merlin Strain, human herpesvirus
6A, human herpesvirus 6B, human herpesvirus 7, human herpes-virus 8
type M, human herpesvirus 8 type P and Human Cytomegalovirus),
human immunodeficiency viruses (HIV) (such as HIV 1 and HIV 2),
human metapneumoviruses, human papillomaviruses, human
parainfluenza viruses (such as human parainfluenza virus 1-3),
human parechoviruses, human parvoviruses (such as human parvovirus
4 and human parvovirus B19), human respiratory syncytial viruses,
human rhinoviruses (such as human rhinovirus A and human rhinovirus
B), human spumaretroviruses, human T-lymphotropic viruses (such as
human T-lymphotropic virus 1 and human T-lymphotropic virus 2),
Human polyoma viruses, Hypoviruses, Leviviruses, Luteoviruses,
Lymphocytic choriomeningitis viruses (LCM), Marnaviruses,
Narnaviruses, Nidovirales, Nodaviruses, Orthomyxoviruses (such as
Influenza viruses), Partitiviruses, Paramyxoviruses (such as
Measles virus and Mumps virus), Picornaviruses (such as Poliovirus,
the common cold virus, and Hepatitis A virus), Potyviruses,
Poxviruses (such as Variola and Cowpox), Sequiviruses, Reoviruses
(such as Rotavirus), Rhabdoviruses (such as Rabies virus),
Rhabdoviruses (such as Vesicular stomatitis virus, Tetraviruses,
Togaviruses (such as Rubella virus and Ross River virus),
Tombusviruses, Totiviruses, Tymoviruses, and Noroviruses among
others.
[0111] Viral antigens may be from a Hepatitis C virus (HCV). HCV
antigens may be selected from one or more of E1, E2, E1/E2, NS345
polyprotein, NS 345-core polyprotein, core, and/or peptides from
the nonstructural regions (Houghton et al. (1991) Hepatology
14:381-388, which is incorporated by reference).
[0112] Viral antigens may be derived from a Human Herpes virus,
such as Herpes Simplex Virus (HSV), Varicella-zoster virus (VZV),
Epstein-Barr virus (EBV), or Cytomegalovirus (CMV). Human Herpes
virus antigens may be selected from immediate early proteins, early
proteins, and late proteins. HSV antigens may be derived from HSV-I
or HSV-2 strains. HSV antigens may be selected from glycoproteins
gB, gC, gD and gH, or immune escape proteins (gC, gE, or gl). VZV
antigens may be selected from core, nucleocapsid, tegument, or
envelope proteins. A live attenuated VZV vaccine is commercially
available. EBV antigens may be selected from early antigen (EA)
proteins, viral capsid antigen (VCA), and glycoproteins of the
membrane antigen (MA). CMV antigens may be selected from capsid
proteins, envelope glycoproteins (such as gB and gH), and tegument
proteins. Exemplary herpes antigens include (GENBANK.TM. Accession
No. in parentheses) those derived from human herpesvirus 1 (Herpes
simplex virus type 1) (NC 001806), human herpesvirus 2 (Herpes
simplex virus type 2) (NC 001798), human herpesvirus 3 (Varicella
zoster virus) (NC 001348), human herpesvirus 4 type 1 (Epstein-Barr
virus type 1) (NC 007605), human herpesvirus 4 type 2 (Epstein-Ban
virus type 2) (NC 009334), human herpesvirus 5 strain AD169 (NC
001347), human herpesvirus 5 strain Merlin Strain (NC 006273),
human herpesvirus 6A (NC 001664), human herpesvirus 6B (NC 000898),
human herpesvirus 7 (NC 001716), human herpesvirus 8 type M (NC
003409), and human herpesvirus 8 type P (NC 009333).
[0113] Human Papilloma virus (HPV) antigens are known in the art
and can be found for example in International Patent Publication
No. WO96/19496, (incorporated by reference in its entirety) which
discloses variants of HPV E6 and E7 proteins, particularly fusion
proteins of E6/E7 with a deletion in both the E6 and E7 proteins.
HPV L1 based antigens are disclosed in international Patent
publication Nos. WO94/00152, WO94/20137, WO93/02184 and WO94/05792,
all of which are incorporated by reference. Such an antigen can
include the L1 antigen as a monomer, a capsomer or a virus like
particle. Such particles may additionally comprise L2 proteins.
Other HPV antigens are the early proteins, such as E7 or fusion
proteins such as L2-E7. Exemplary HPV antigens include (GENBANK.TM.
Accession No. in parentheses) those derived from human
papillomavirus-1 (NC 001356), human papillomavirus-18 (NC 001357),
human papillomavirus-2 (NC 001352), human papillomavirus-54 (NC
001676), human papillomavirus-61 (NC 001694), human
papillomavirus-cand90 (NC 004104), human papillomavirus RTRX7 (NC
004761), human papillomavirus type 10 (NC 001576), human
papillomavirus type 101 (NC 008189), human papillomavirus type 103
(NC 008188), human papillomavirus type 107 (NC 009239), human
papillomavirus type 16 (NC 001526), human papillomavirus type 24
(NC 001683), human papillomavirus type 26 (NC 001583), human
papillomavirus type 32 (NC 001586), human papillomavirus type 34
(NC 001587), human papillomavirus type 4 (NC 001457), human
papillomavirus type 41 (NC 001354), human papillomavirus type 48
(NC 001690), human papillomavirus type 49 (NC 001591), human
papillomavirus type 5 (NC 001531), human papillomavirus type 50 (NC
001691), human papillomavirus type 53 (NC 001593), human
papillomavirus type 60 (NC 001693), human papillomavirus type 63
(NC 001458), human papillomavirus type 6b (NC 001355), human
papillomavirus type 7 (NC 001595), human papillomavirus type 71 (NC
002644), human papillomavirus type 9 (NC 001596), human
papillomavirus type 92 (NC 004500), and human papillomavirus type
96 (NC 005134).
[0114] Viral antigens may be derived from a Retrovirus, such as an
Oncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may be
derived from HTLV-I, HTLV-2 or HTLV-5. Lentivirus antigens may be
derived from HIV-1 or HIV-2. Retrovirus antigens may be selected
from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr.
Antigens for HIV are known in the art, for example HIV antigens may
be selected from gag (p24gag and p55gag), env (gp160 and gp41),
pol, tat, nef, rev vpu, miniproteins, (p55 gag and gp140v). HIV
antigens may be derived from one or more of the following strains:
HIVmb, HIV; HIVLAV, HIVLAI, HIVM N, HIV-1 CM235, HIV-1 US4.
Examples of HIV antigens can be found in International Patent
Publication Nos. WO 09/089,568, WO 09/080,719, WO 08/099,284, and
WO 00/15255, and U.S. Pat. Nos. 7,531,181 and 6,225,443, all of
which are incorporated by reference. Exemplary HIV antigens include
(GEN-BANK.TM. Accession No. in parentheses) those derived from
human immunodeficiency virus 1 (NC 001802), human immunodeficiency
virus 2 (NC 001722).
[0115] In some embodiments, a disclosed functionalized electrode
includes one or more antigens derived from one or more of the
viruses listed above. In some embodiments, an antibody that
specifically binds antigens derived from one or more of the viruses
listed above is part of a functionalized electrode. Thus in some
examples the disclosed functionalized electrodes can be used to
detect such viruses in a sample, for example to diagnose a viral
infection or the presence of a virus in an environmental
sample.
[0116] The general performance of electrochemical sensors is often
determined by the surface architectures that connect the sensing
element to the biological sample at the nanometer scale.
Electrochemical biosensors have suffered from a lack of surface
architectures allowing high enough sensitivity and unique
identification of the response with the desired biochemical
event.
[0117] Various prior attempts have been made to fashion biosensors
out of long chain self-assembled monolayers (SAMs) because of the
desirable characteristics of self-assembled monolayers, such as
stability and resistance to non-specific biomolecule adsorption.
However, electrochemical sensors based on long chain alkyls have
suffered from limited applicability because of their low
permeability to electron transfer (see e.g. Fragoso et al., Anal.
Chem., 80:2556-2563, 2008). In an attempt to overcome the perceived
limitations present in long chain SAMs, Fragoso et al. turned to
dithiols, which are believed to be less insulating. However, one of
the advantages of using long chain SAMs is lost by turning to a
less insulating monolayer, namely the loss of selectivity against
non-specific electron transfer, which reduces the signal to noise
of the sensor and therefore the sensitivity.
[0118] Another drawback to the use of SAMs is that they are ionic
insulators, that is ions are not readily able to penetrate SAM in
order to transfer electrons to and from the underlying
electro-conductive material of an electrochemical sensor (see e.g.
Boubour and Lennox, Langmuir 16:42224228, 2000). While the
insulating properties of SAMs are desirable from the standpoint of
limiting non-specific electron transfer, in the absence of
selective ionic transfer for an analyte of interest, SAMs have
limited use as components of electrochemical sensors.
[0119] As disclosed herein, the limitations present in previous
attempts to create sensors from long chain thiol containing SAMs
have been overcome by careful selection of thiol or dithiol
compounds that retain their insulating properties toward
non-specific electron transfer coupled with the selection of enzyme
reaction products that are electro-active and capable of
facilitating electron transfer through the monolayer to the
electron conducting surface. Thus, disclosed herein it has been
surprisingly found that functionalized electrodes can be formed
that retain the beneficial insulating properties on SAMs such as to
yield high signal to noise, in conjunction with high selectivity
and sensitivity.
[0120] Kits are also provided herein. Kits for detecting analytes
of interest contain a one or more of the disclosed biosensors. In
some embodiments, a kit includes instructional materials disclosing
means of detecting analytes of interest. The instructional
materials may be written, in an electronic form (such as a computer
diskette or compact disk) or may be visual (such as video files).
The kits may also contain detection reagents and substrates that
have electro-active reaction product. The kits may also include
additional components to facilitate the particular application for
which the kit is designed. Thus, for example, the kit may
additionally contain buffers and other reagents routinely used for
the practice of a particular method. Such kits and appropriate
contents are well known to those of skill in the art. In some
examples the kits contain controls, for examples control solutions
containing a known amount or concentration of a target analyte, for
example as a means calibrate the biosensors included in kits. The
kit may contain components for automated assay testing, and
automated data collection that would be useful in a rapid,
point-of-care setting.
[0121] Preferred embodiments of the method according to this
invention are those wherein: [0122] (A) the cross-linker compound
is a compound of formula (I),
[0122] HS--(CH.sub.2).sub.m--CH(ZH)-SPACER-(CH.sub.2).sub.p-A (I)
[0123] in which [0124] m is an integer from 2 to 6, in particular 2
to 4, [0125] A is selected from --CH.dbd.CH.sub.2 and --C.ident.CH,
in particular --CH.dbd.CH.sub.2 and [0126] Z is S or a single bond,
in particular S, [0127] SPACER is a group of formula
[0127]
--(CH.sub.2).sub.n--(C.dbd.O).sub.x--Y--(CH.sub.2CH.sub.2--O).sub-
.y--(CH.sub.2).sub.r(C.dbd.O).sub.v--X-- [0128] wherein [0129] X
and Y are each independently NH or 0, [0130] n is 0 or an integer
from 1 to 10, in particular 2 to 6, most preferably 4, [0131] x and
v are each independently 0 or 1, in particular 1, [0132] y is an
integer from 1 to 20, in particular 4 to 18, [0133] r and p are
each independently selected from an integer from 1 to 6, in
particular 1;
[0134] The polyethylene glycol group (PEG)
--(CH.sub.2CH.sub.2--O).sub.y-- contains either a defined number y
of ethylene oxide units, in particular 4 to 18 units, most
preferred 8 to 16 units, or contains an undefined number of
ethylene oxide units with an average molecular mass of 0.5 to 10
kDa, in particular 2 to 8 kDa, most preferred about 5 kDa; [0135]
(B) the biomolecule is an antibody, an enzyme or nucleic acid, in
particular a monoclonal antibody; [0136] (C) the photo-initiator is
al-benzoyl-1-methyl-ethanol derivative, in particular a
1-benzoyl-1-methyl-ethanol derivative, which is soluble in water,
most preferably 2-hydroxy-4-(2-hydroxyethoxy)-2-metylpropiophenon;
[0137] (D) the irradiation is carried out at a wavelength
.lamda..sub.max of 300 to 340 nm, in particular at about 320
nm.
[0138] Furthermore, the invention relates to the novel compounds of
formula (I), preferred compounds of formula (I) are the following
compounds of formula (IA):
HS--(CH.sub.2).sub.m--CH(SH)-SPACER-CH.sub.2-A (IA)
in which A and SPACER have the meaning given for formula (I) and m
is an integer from 2 to 4, in particular 2, The SPACER of formula
(IA) is preferably a group of formula
--(CH.sub.2).sub.n--(C.dbd.O)--NH--(CH.sub.2CH.sub.2--O).sub.y--CH.sub.2-
--(C.dbd.O)--NH--
wherein n is 0 or an integer from 1 to 10, in particular 2 to 6,
most preferably 4, y is an integer from 1 to 20. Most preferred are
the compounds of formula (IB), which are obtainable from liponic
acid, in particular from R-.alpha.-liponic acid by reduction of the
--S--S-- bond:
HS--(CH.sub.2).sub.2--CH(SH)--(CH.sub.2).sub.4--(C.dbd.O)--NH--(CH.sub.2-
CH.sub.2--O).sub.y--CH.sub.2--(C.dbd.O)--NH--CH.sub.2-A (IB),
wherein A and y have the meaning given for formula (I).
[0139] The preferred compounds of formulae (IA) and (B) can be
synthesized according to the following Reaction Scheme I:
##STR00002##
[0140] In Step 1) and Step 2) the corresponding acids are treated
with an alcohol (Y or X.dbd.O) or an amine (Y or X.dbd.NH) under
conditions of an esterification reaction or an amide forming
reaction. Such reaction conditions are well known for the person
skilled in the art. Either the acid is activated for example by
treatment with N-hydroxysuccinimide (NHS) or thionyl chloride
and/or the water formed during the reaction is irreversibly trapped
by a dehydration agent.
[0141] Most preferred the acid is treated with the alcohol or amine
in the presence of N-hydroxysuccinimide (NHS), sodium
N-hydroxysulfosuccinimide, 1-hydroxybenzotriazol (HOBT),
1-hydroxy-7-azabenzotriazole (HOAT) or pentafluorphenol and
dicyclohexylcarbodiimide (DCC) or
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
[0142] In Step 3) the disulfide bond is reductively cleaved by
treatment with an reduction agent, in particular with DTT
(dithiothreitol), mercaptoethanol, tris(2-carboxyethyl)phosphine
(TCEP) and/or DTE (dithioerythritol).
[0143] The preferred compounds of formula I, wherein X is S and v
is 0 can be prepared according to the following Reaction Scheme
II:
##STR00003##
[0144] The protecting group PG is a group suitable to protect amino
or hydroxyl groups. Such protecting groups and the methods of
deprotection (Step 2)) are well known to the person skilled in the
art, cp.: T. W. Green, P. G. M. Wuts, Protective Groups in Organic
Synthesis, Wiley-Interscience, New York, 1999. Most preferred
protecting groups for amino groups are for example:
9-Fluorenyl-methyl carbamate, (FMOC amino), t-butyl carbamate (BOC
amino), benzyl carbamate, acetamide, trifluoroacetamide,
benzylamine and tritylamine. Most preferred protecting groups for
hydroxy groups are for example: methoxymethyl ether,
tetrahydropyranyl ether, t-butyl ether, benzyl ether,
trihydrocarbylsilylether such as t-butyldimethylsilyl ether or
t-butyldiphenylsilyl ether.
[0145] The substitution reaction in Step 3) is as rule carried out
in the presence of a base such as alkali metal hydroxide,
preferably sodium hydroxide or potassium hydroxide, or an tertiary
amine, preferably triethylamine or
2,2,6,6-tetrametylpiperidine.
[0146] Step 4 can be carried out analogously as described for
Reaction Scheme 1, Step 3).
[0147] Accordingly, the new liponic acid derivatives of formula
(II) are another subject matter of the present invention as
intermediates for the preparation of the compounds of formulae (I),
(IA) and (IB).
[0148] Most preferred are the compounds of formula (IIA),
##STR00004##
in particular the compounds of formula (IIA1),
##STR00005##
wherein SPACER, A and p have the meaning given for formula (I).
[0149] The present disclosure shall be illustrated by the following
non-limiting Examples.
EXAMPLES
Abbreviations
[0150] AcOH acetic acid approx. approximately .degree. C. degrees
Celsius DCC dicyclohexylcarbodiimide DCM dichloromethane DTT
dithiothreitol kDa kilodalton MeCN acetonitrile MeOH methanol mL
milliliter mM millimolar
NHS N-hydroxysuccinimide
[0151] PEG-alkene methoxy poly (ethylene glycol) alkene PEG-alkyne
methoxy poly (ethylene glycol) alkyne rpm revolutions per min
.lamda. wavelength TCEP tris(2-carboxyethyl)phosphine TEA
triethylamine TFA trifluoroacetic acid
Example 1
[0152] Synthesis of SH-PEG-Alkene/-Alkyne Cross-Linker
[0153] For coupling of diagnostic biomolecules to microchips via
PEG-alkene/-alkyne cross-linker PEG-alkene and PEG-alkyne molecules
with average molecular weights of 800 Da and 5000 Da were
synthesized. Furthermore, a SH-mPEG of 600 Da that was not
functionalized with alkene or alkyne has been synthesized. This
molecule can be used for dilution of the PEG-alkene/-alkyne
cross-linker during coating of the metal surface in order to obtain
a homogenous statistical distribution of functional groups on the
chip surface.
Experimental
[0154] RP-HPLC--
[0155] RP-HPLC analysis was performed on a Water alliance system
equipped with an ELSD detector and a photo diode array detector. An
XBRIDGE.TM. BEH300 4.6.times.250 mm 5 .mu.m RP18 column was used.
HPLC-H.sub.2O (+0.1% TFA) and HPLC-MeCN (+0.1% TFA) were used as
eluents in a gradient HPLC.
[0156] NMR--
[0157] For NMR spectroscopy 30-60 mg of the analyte were solved in
600 .mu.L CDCl3 and transferred to an NMR vial. The spectroscopy
was performed using a Varian Mercury-400BB at (1H, 400 MHz; 13C,
100.6 MHz) or a Varian Mercury-300BB (1H, 300 MHz; 13C, 75.5 MHz).
The chemical shift is indicated in ppm and was calibrated to the
solvent signal.
[0158] MALDI-TOF-MS--
[0159] MALDI-TOF-MS analysis was performed in the linear mode using
an .alpha.-cyano-4-hydroxy-cinnamic acid (CHCA) matrix on an Axima
confidence system (Shimadzu).
[0160] Stirring--
[0161] A magnetic stir bar were added to all reaction mixtures and
crystallization suspensions to stir them with a MR3001 K (Heidolph)
magnetic stirrer.
[0162] Evaporation and Drying--
[0163] Solvents were removed under reduced pressure to dryness with
a Laborota 4000-efficient (Heidolph) rotary evaporator with a
Vacuum Pump Unit PC510 (Vacuubrand) and a water bath temperature at
40.degree. C. The same procedure is used for drying materials.
[0164] Chromatography--
[0165] Chromatography was performed using 40-63 .mu.m silica gel
packed in glass columns with a mixture of different typical organic
solvents. The fractions were collected manually and analyzed by
TLC. The pure product containing fractions were combined and the
solvent was removed under reduced pressure.
[0166] TLC--
[0167] TLC analysis was carried out on TLC Silica gel 60 F254
aluminium sheets with different mixtures of typical organic
solvents. An aqueous potassium permanganate solution was used as
staining reagent.
Example 1.1
[0168] Synthesis of R-.alpha.-Lipoic Acid-PEG12-Allyl (B)
##STR00006##
[0169] Step 1:
[0170] To a solution of R-.alpha.-lipoic acid (668 mg, 3.24 mmol)
in 40 mL DCM, DCC (1.00 g, 4.86 mmol) and NHS (599 mg, 4.86 mmol)
were added and the resulting reaction mixture was stirred for 1 h
at room temperature. The precipitated dicyclohexylurea was removed
by filtration and washed twice with 10 mL DCM. To the resulting
clear yellow reaction mixture NH.sub.2-PEG12-COOH (2.00 g, 3.24
mmol) and TEA (900 .mu.L, 6.48 mmol) were added. The reaction
mixture was stirred for 2.5 h at room temperature. Subsequently the
solvent was removed under reduced pressure. After purification of
the resulting remains by chromatography (35 g silica gel (40-63
.mu.m DCM/MeOH-98:2->90:10, +0.1% AcOH) 1.99 g referring to a
yield of 76% of the product (A) were obtained. According to RP-HPLC
analysis the product contained approx. 3.3% lipoic acid and not
identified impurity that amounted to 2.8%. The overall purity was
approx. 94%.
[0171] Step 2:
[0172] To a solution of the product 0 obtained in Step 1 (1.20 g,
1.49 mmol) in 20 mL DCM, DCC (461 mg, 2.23 mmol) and NHS (257 mg,
2.23 mmol) were added and the resulting reaction mixture was
stirred for 1 h at room temperature. The precipitated
dicyclohexylurea was removed by filtration and washed twice with 5
mL DCM. To the resulting clear yellow reaction mixture allylamine
(224 .mu.L, 2.98 mmol) and TEA (415 .mu.L, 2.99 mmol) were added.
The reaction mixture was stirred for 17 h at room temperature and
subsequently washed with 20 mL water. The organic phase was
concentrated under reduced pressure and the resulting solid was
purified by chromatography (15 g silica gel (40-63 .mu.m
DCM/MeOH-98:2->95:5). 796 mg referring to a yield of 63% of the
product CES0594 were obtained. According to RP-HPLC analysis (FIG.
2) the product had a purity of >99%. The molecular weight and
identity of (B) were confirmed by MALDI-MS and NMR.
Example 1.2
[0173] Synthesis of R-.alpha.-Lipoic-Acid-PEG12-Propargyl (C)
##STR00007##
[0174] To a solution of product (A) as obtained in Step 1 of
example 1.1. (1.52 g, 1.89 mmol) in 40 mL DCM, DCC (592 mg, 2.87
mmol) and NHS (325 mg, 2.82 mmol) were added and the resulting
reaction mixture was stirred for 1 h at room temperature. The
precipitated dicyclohexylurea was removed by filtration and washed
twice with 5 mL DCM.
[0175] To the resulting clear yellow reaction mixture
propargylamine (245 .mu.L, 3.82 mmol) and TEA (525 .mu.L, 3.79
mmol) were added. The reaction mixture was stirred for 1 h at room
temperature and subsequently washed with 20 mL water. The organic
phase was concentrated under reduced pressure and the resulting
solid was purified by chromatography (21 g silica gel (40-63 .mu.m
DCM/MeOH-98:2->95:5). 1.01 g referring to a yield of 64% of the
product CES0596 were obtained. According to RP-HPLC analysis the
product contained no detectable impurities. The molecular weight
and identity of (C) were confirmed by MALDI-MS and NMR
Example 1.3
[0176] Synthesis of R-.alpha.-Lipoic-Acid-mPEG8 (D)
##STR00008##
[0177] To a solution of R-.alpha.-lipoic-acid (538 mg, 2.61 mmol)
in 40 mL DCM, DCC (8.11 mg, 3.93 mmol) and NHS (450 mg, 3.91 mmol)
were added and the resulting reaction mixture was stirred for 1 h
at room temperature. The precipitated dicyclohexylurea was removed
by filtration and washed twice with 5 mL DCM. To the resulting
clear yellow reaction mixture NH2-mPEG8 (0.98 g, 2.56 mmol) and TEA
(725 .mu.L, 5.23 mmol) were added. The reaction mixture was stirred
for 1 h at room temperature and subsequently washed with 30 mL
water. The organic phase was concentrated under reduced pressure
and the resulting solid was purified by chromatography (34 g silica
gel (40-63 .mu.m) DCM/MeOH-98:2->97:3). 1.12 g referring to a
yield of 75% of the product (D) were obtained. According to RP-HPLC
analysis the product had a purity of approx. 99.8%. The molecular
weight and identity of CES0598 were confirmed by MALDI-MS and
NMR
Example 1.4
[0178] Synthesis of R-.alpha.-Lipoic-Acid-5 kDa PEG-Allyl (F)
##STR00009##
[0179] Step 1:
[0180] To a solution R-.alpha.-lipoic-acid (125 mg, 606 .mu.mol) in
5 mL DCM, DCC (184 mg, 892 .mu.mol) and NHS (108 mg, 938 .mu.mol)
were added and the resulting reaction mixture was stirred for 1 h
at room temperature. The precipitated dicyclohexylurea was removed
by filtration and washed twice with 2 mL DCM. To the resulting
clear yellow reaction mixture NH2-5 kDa PEG-COOH (3.03 g, 606
.mu.mol) and TEA (166 .mu.L, 1198 .mu.L) in DCM (91 mL) were added.
The reaction mixture was stirred for 17 h at room temperature and
subsequently washed with 50 mL 100 mM citric acid. The organic
phase was concentrated under reduced pressure and the resulting
solid matter was purified by chromatography (36 g silica gel (40-63
.mu.m DCM/MeOH 98:2->90:10+0.1% AcOH). 2.75 g referring to a
yield of 88% of the product (E) were obtained. According to RP-HPLC
analysis, the product had a purity of approx. 80% with 20% of
unknown impurities. These impurities were removed during the
purification of the next step.
[0181] Step 2:
[0182] To a solution of (E) (1.30 g, 251 .mu.mol) in 40 mL DCM, DCC
(76 mg, 368 .mu.mol) and NHS (42 mg, 365 .mu.mol) were added and
the resulting reaction mixture was stirred for 6 h at room
temperature. To the yellow slightly turbid solution allylamine (50
.mu.L, 666 .mu.mol) and TEA (100 .mu.L, 721 .mu.mol) were added and
the mixture was stirred for 18 hrs. at room temperature. The
precipitated dicyclohexylurea and free N-hydroxysuccinimide were
removed by filtration and washed twice with 10 mL DCM. The reaction
mixture was washed with 30 mL water. The organic phase was
concentrated under reduced pressure and the resulting solid was
purified by chromatography (31 g silica gel (40-63 .mu.m
DCM/MeOH-95:5->80:20). 1.18 g referring to a yield of 90% of the
product (F) were obtained. According to RP-HPLC analysis the
product had a purity of approx. 97.5%. The molecular weight and
identity of (F) were confirmed by MALDI-MS and NMR.
Example 1.5
[0183] Synthesis of R-.alpha.-Lipoic-Acid-5 kDa PEG-Propargyl
(G)
##STR00010##
[0184] To a solution of product (E) obtained according to Step 1 of
Example 1.4 (1.30 g, 251 .mu.mol) in 40 mL DCM, DCC (116 mg, 562
.mu.mol) and NHS (59 mg, 513 mmol) were added and the resulting
reaction mixture was stirred for 3.5 hrs. at room temperature. To
the yellow slightly turbid solution propargylamine (48 .mu.L, 749
.mu.mol) and TEA (104 .mu.L, 750 .mu.mol) were added and the
mixture was stirred for 20 hrs. at room temperature. The
precipitated dicyclohexylurea and free N-hydroxysuccinimide were
removed by filtration and washed twice with 10 mL DCM. The reaction
mixture was washed with 30 mL water. The organic phase was
concentrated under reduced pressure and the resulting solid matter
was purified by chromatography (32 g silica gel (40-63 .mu.m
DCM/MeOH-95:5->80:20). 1.18 g referring to a yield of 90% of the
product (G) were obtained. According to RP-HPLC analysis the
product had a purity of approx. 95%. The molecular weight and
identity of (G) were confirmed by MALDI-MS and NMR.
Example 2
Example 2.1. Investigation of the Photochemical Coupling
Conditions
[0185] A model system was used to establish the photochemical
reaction of R-.alpha.-lipoic-acid-PEG12-allyl (B) with 3-mercapto
propionic acid (Scheme 7):
##STR00011##
[0186] The optimized conditions were used to investigate the
photochemical modification of a partially reduced antibody.
[0187] All experiments were performed using only photo initiator I
(2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) since photo
initiator II (2,2-Dimethoxy-2-phenyphenone) showed insufficient
solubility in aqueous solutions (<1 mg/mL 10% DMSOaq). In
contrast, photo initiator I was soluble in water up to a
concentration of 1 mg/mL without addition of DMSO.
[0188] In initial experiments, a stock solution of product (B) of 1
mg/ml in water was treated with 10 mol % photo initiator I. The
reaction mixture was separated into 5 tubes (200 .mu.l per tube,
A-E). To these tubes different molar excesses of 3-mercapto
propionic acid (SH) were added (see Table 1, entry A to E).
TABLE-US-00001 Tube Eq. SH A 5 B 25 C 50 D 100 E 250
[0189] The reaction was induced using the following conditions:
[0190] Reaction temperature=1.degree. C.
[0191] Light exposure time=30 min
[0192] .lamda..sub.max: 310 nm
[0193] Immediately after irradiation samples were analyzed by HPLC.
In summary, these experiments demonstrated feasibility of the photo
initiated coupling of thiol reagents to the alkyne group of the
synthesized cross-linker.
[0194] Further experiments were performed to determine velocity of
the reaction and dependency on the reaction volume. During a period
of 300 seconds approx. 60% of conversion was obtained and a linear
regression described the reaction process well. Conversion did not
depend on the applied reaction volume. Reaction volumes of 20 .mu.L
or 200 .mu.L resulted in the same conversion in the same reaction
time. In contrast, typical kinetics of such reaction types
(Macromol. Rapid Commun. 2010, 31, 1247-1266) show a logarithmic
correlation. This feasibility study focused on the reproducibility
of this photo induced click reaction rather than full conversion.
In this regard, a reproducible conversion of 2% was already
obtained after 5 seconds.
[0195] Furthermore, based on these results the first experiments
with a reduced antibody were designed. For reaction development
only the 5 kDa PEG conjugates (alkene (F) and alkyne (G)) were
used, as due to the mass difference the respective reaction
products could be detected by SDS-PAGE.
Example 3
3.1. Partial Reduction of Antibodies
[0196] Free thiol groups can be introduced to antibodies by partial
reduction of the antibody's internal disulfide bonds. Therefore
reducing conditions should be investigated that result in partial
reduction of the antibody without affecting its activity. Reactions
should be performed in a volume of 25-50 .mu.L. DTT, TCEP and
cysteamine should be tested as reducing agent. Partially reduced
antibody should then be modified with 5 kDa mPEG-Maleimide, which
reacts with free sulfhydryl groups to investigate the success of
the reduction reaction.
3.2. Experimental
[0197] Reducing Agent Screening--
[0198] The reducing agent screening experiments were performed at
protein concentrations of 0.5 mg/mL (anti-ACTH antibody) or 0.5, 5,
10 and 15 mg/mL (IgG1) using varying reducing agents excesses as
indicated in the results section. The reactions were performed at
room temperature for 1 or 2 h in final volume of 25 .mu.L using 20
mM Na phosphate pH 7.2 as reaction buffer.
[0199] PEGylation of the Antibodies--
[0200] The PEGylation reaction was performed at room temperature
for 3 h in 20 mM Na phosphate pH 7.2 at a molar PEG: protein ratio
of 400:1. Protein concentrations in the reaction mixture ranged
from 0.3 mg/mL (anti-ACTH antibody) to 0.3, 3, 6 and 9 mg/mL
(IgG1).
[0201] SDS-PAGE--
[0202] SDS-PAGE was performed under reducing or non-reducing
conditions, using 4-12% Bis-Tris gels of the NuPAGE system from
Invitrogen. Gels were stained with SIMPLYBLUE.TM. SafeStain kit
(Invitrogen).
[0203] Size Exclusion Chromatography--
[0204] SEC was performed on an Agilent 1200 using Superdex 200
10/300. SEC was performed at a flow rate of 0.65 mL/min. using PBS
(10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.7
mM KCl, pH 7.4) as eluent.
3.2. Results
[0205] In initial experiments partial reduction of an anti-ACTH
antibody was investigated using different concentrations of the
reducing agents. In this way the concentration of the reducing
agents should be determined at which complete, undissociated IgG
can be obtained.
[0206] A generic murine IgG1 antibody (Biogenes) was used to
investigate suitable conditions for partial reduction in more
detail.
[0207] In particular, the impact of the IgG1 concentration on
reduction efficacy was investigated for DTT. 5 kDa mPEG-Maleimide
was added at high excess (400-fold) to reaction mixtures in order
to detect all generated free thiol groups quantitatively as a band
shift in SDS-PAGE.
[0208] Generally, with increasing excess of reducing agent higher
ratios of reduced IgG1 were detected. Additionally, at higher
protein concentrations lower DTT excesses were required to induce
reduction of IgG1. Using a 6 or 10 eq. DTT, 0-5-fold modification
of the heavy chain with 5 kDa mPEG-Maleimide was obtained according
to SDS-PAGE analysis. Reduction with 1.5 or 3 eq. DTT resulted
mainly in mono-PEGylation of the heavy chain, however, higher
degrees of modification were also observed to a lesser extent. Only
mono-modification of the light chain was observed at ratios of 1%
(1.5 eq. DTT) to approx. 20% (10 eq. DTT).
[0209] Furthermore, the more IgG1 was in the reduced form, the
higher the degree of PEGylation. The impact of disulfide reduction
on the assembly state of IgG1 after addition of reducing agent and
PEG was further analyzed by SEC
[0210] Non-reduced IgG1 eluted at 20.1 min as a single and regular
peak. Treatment with a 10-fold excess of DTT had no detectable
effect on retention time or profile of the antibody indicating
integrity under physiological conditions. This finding was in
contrast to the findings by SDS-PAGE in which dissociation of IgG1
into heavy and light was observed under the same conditions.
[0211] Modification of the partially reduced IgG1 (10-fold excess
of DTT) with 5 kDa mPEG-Mal resulted in broadening of the peak to
lower retention times between 14.5 and 22 min. This was expected
for a full antibody modified with 5 kDa mPEG. Signals at higher
retention times that would have indicated dissociation of the
antibody were not observed.
[0212] At lower DTT excess (1.5-fold), the peak obtained during SEC
was less broad compared to 10-fold DTT excess, indicating a lower
degree of PEGylation.
[0213] The impact of the reducing time was investigated for all
three reducing agents at varying molar excesses using anti-ACTH
antibody. After 1 h and 2 h of incubation reduction was stopped by
addition of a high molar excess of 5 kDa mPEG-Maleimide.
[0214] Under all investigated conditions whole anti-ACTH antibody
was observed by SDS-PAGE, indicating incomplete reduction of the
interchain disulfide bonds. However, at increasing excesses of DTT
or TCEP higher ratios of heavy and light chain as well as a higher
degree of PEGylation were detected. The effect was slightly
stronger at 2 h reducing time. Using cysteamine no notable
dissociation of anti-ACTH antibody at 300-900-fold excess of
reducing agent could be obtained. Based on these results it was
decided to use 1 h reducing time. Furthermore, for preparation of
PEGylated anti-ACTH antibody TCEP was chosen as reducing agent, as
it is not modified with Maleimide activated molecules.
Example 4
Example 4.1. Coupling of PEG-Alkene/-Alkyne to Partially Reduced
Antibodies by Photoreaction
[0215] Photoreaction conditions for the coupling of antibodies to
the alkene/alkyne function of the PEG-linker (F) and (G) have been
developed. Therefore, different reaction conditions like light
exposure time, PEG-excess, photo initiator I ("Photo I")
concentration, protein concentration and reaction pH value shall be
investigated. The coupling efficiency has been analyzed by SDS-PAGE
of the resulting conjugates.
4.2 Experimental
[0216] (Partial) Reduction of Antibodies--
[0217] To an antibody solution with a protein concentration of 15
mg TCEP was added at a final concentration between 0.01 and 1 mM,
corresponding to a molar excess of 0.1 and 10-fold over the
antibody. The reducing reaction was performed in 20 mM Na phosphate
pH 7.2 for 1 h at room temperature. Subsequently the antibody was
modified with PEG-alkene or PEG-alkyne by photo click
chemistry.
[0218] Modification of Antibodies with PEG-Alkene/-Alkyne by Photo
Click Chemistry--
[0219] Modification of the antibodies with PEG-alkene lot CES0601
or PEG-alkyne lot CES0602 was performed by photo induction using a
CAPROBOX.TM. (Caprotech) under varying conditions as described in
the following section.
4.3. Results
[0220] For initial photochemical coupling experiments of the
PEG-alkenes the commercially available antibody IgG1 (mouse) was
used. The antibody was reduced with 1 mM TCEP (10 eq.) at a protein
concentration of 15 mg/mL for 1 h. The impact of PEG-excess on
antibody modification was tested with reduced IgG1 using the
following conditions:
[0221] IgG1 concentration: 1 mg/mL
[0222] Reaction buffer: 20 mM Na phosphate, pH 7.2
[0223] Molar PEG-alkene (F) excesses over IgG1: 0.2- to 10-fold
[0224] Photoinitiator I excess: 0.1 mol/mol PEG
[0225] Light exposure time: 30 min.
PEGylation of IgG1 was monitored by SDS-PAGE analysis
[0226] By SDS-PAGE two bands at 55 kDa and 25 kDa were detected for
the reduced but not PEGylated IgG1, which were assigned to the
heavy and light chain respectively. For all samples conjugated with
PEG-alkene by photo induction several high molecular weight bands
were observed. These bands probably correspond to aggregated or
incorrectly assembled protein as they did not match the expected
molecular weight difference of approx. 10 kDa for a PEGylated
antibody fragment. Only at a PEG-excess of 10 eq. a band at approx.
65 kDa was observed, which was assigned to the mono-PEGylated heavy
chain. For an IgG1 sample that contained PEG and photo initiator
but was not exposed to light, no additional bands aside from the
heavy and light chain were detected. This indicates that
aggregation was caused by light exposure.
[0227] As PEGylated heavy chain was obtained, it was assumed that
modification of the antibody by photo click chemistry is feasible
but the reaction had to be optimized towards reduction of
aggregated antibody.
[0228] In order to reduce protein aggregation and increase
PEGylation different light exposure times and photo initiator I
concentrations were investigated. Therefore IgG1 reduced with TCEP
was modified with PEG-alkene using the following conditions:
[0229] IgG1 concentration: 1 mg/mL
[0230] Reaction buffer: 20 mM Na phosphate, pH 7.2
[0231] Molar PEG-alkene excess over IgG1: 10-fold
[0232] Photo initiator I excess: 1-10 mol/mol PEG
[0233] Light exposure time: 5 s-10 min
[0234] Increase of photo initiator I excess resulted in higher
fractions of aggregates at all light exposure times, whereas the
ratio of mono-PEGylated heavy chain was not affected. By reduction
of light exposure to 5-30 seconds the fraction of aggregates could
notably be reduced, however at this time range also the fraction of
mono-PEGylated heavy chain decreased with the exposure time.
[0235] In order to minimize a possible incorrect assembly via
interchain disulfide formation, it was decided to reduce the excess
of TCEP over IgG1 to 1 or 5 eq. during the initial antibody
reduction. Subsequently the antibody was modified with 1 eq.
PEG-alkene using 1 eq. photo initiator I at different light
exposure times
[0236] By reduction of the TCEP excess, the ratio of aggregates
could be decreased towards approx. 9% using 1 eq. TCEP at a light
exposure time of 10 min. However, as a short light exposure is
aspired for the final chip coating process, it was decided to
choose a standard time of 0.5 min, which resulted in 13%
mono-PEGylated heavy chain at 1-fold and 15% at 5-fold TCEP excess.
Furthermore approx. 1% and 3% mono-PEGylated light chain were
detected at 1 and
[0237] 5 eq. TCEP, respectively. As at 1 eq. TCEP a notably lower
fraction of aggregates was detected, it was decided to choose it as
the standard TCEP excess.
[0238] The PEG-excess was further optimized for the standard light
exposure time using the following conditions:
[0239] IgG1 concentration: 1 mg/mL
[0240] Reaction buffer: 20 mM Na phosphate, pH 7.2
[0241] Molar PEG-alkene excesses over IgG1: 0.5-10-fold
[0242] Photo initiator I excess: 1 mol/mol PEG
[0243] Light exposure time: 30 s
[0244] With increasing PEG-excess a higher fraction of
mono-PEGylated heavy chain was observed.
[0245] The highest ratio (7%) of mono-PEGylated heavy chain was
obtained at 10 eq. PEG, which was therefore chosen as the standard
PEG-excess.
[0246] The protein concentration in the reaction mixture was
optimized using the following reaction parameters:
[0247] IgG1 concentration: 0.2-10-mg/mL
[0248] Reaction buffer: 20 mM Na phosphate, pH 7.2
[0249] Molar PEG-alkene excess over IgG1: 10-fold
[0250] Photo initiator I excess: 1 mol/mol PEG
[0251] Light exposure time: 30 s
[0252] The fraction of mono-PEGylated heavy chain increased at
higher protein concentrations. However, the fraction of high
molecular weight impurities (aggregates and multiply PEGylated
antibody) increased as well.
[0253] The highest fraction of mono-PEGylated heavy chain (16%) was
detected at a protein concentration of 2 mg/mL. However, in order
to obtain a high protein concentration, the antibody has to be
concentrated in a previous step. For the aspired coating of the
chip the antibody, the concentrations of the antibodies and PEG:
protein ratios will have to be further optimized. Therefore for the
reaction in solution a standard protein concentration of 1 mg/mL
was chosen, in order to minimize pre-concentration of the
antibody.
[0254] The reaction pH value was optimized using the following
reaction parameters:
[0255] IgG1 concentration: 1 mg/mL
[0256] Reaction buffers and pH values: 100 mM Na acetate, pH 5.0;
20 mM Na phosphate pH 7.2; 100 mM Na borate, pH 9.0
[0257] Molar PEG-alkene excess over IgG1: 10-fold
[0258] Photo initiator I excess: 1 mol/mol PEG
[0259] Light exposure time: 30 s
[0260] At reaction pH 5, the highest fraction of mono-PEGylated
light chain (2%) was detected, however a low ratio (6%) of
mono-PEGylated heavy chain was obtained. Only a slightly higher
ratio (7%) mono-PEGylated light chain was observed at pH 7. At pH
9, the highest fraction (9%) of mono-PEGylated was detected, but
also the highest ratio of HMWI (22%) was obtained. It was therefore
decided to use a standard reaction pH value of 7, at which less
(18%) HMWI were detected.
[0261] The following standard reaction parameters were chosen:
Protein concentration: 1 mg/mL
[0262] Reaction buffer: 20 mM Na phosphate pH 7.2
[0263] Molar PEG-alkene/-alkyne excess over IgG1: 10-fold
[0264] Photo initiator I excess: 1 mol/mol PEG
[0265] Light exposure time: 30 s
[0266] The standard reaction parameters were transferred to
PEG-alkyne and modification of anti-ACTH antibody. As reduction of
IgG1 and anti-ACTH antibody was obtained at different reducing
agent in previous experiments, different TCEP concentrations were
investigated.
[0267] For both conjugates (using PEG-alkene or PEG-alkyne) the
highest fraction of mono-PEGylated protein (approx. 7%) was
obtained at the lowest TCEP excess (0.1 eq.), which was therefore
chosen as standard the condition.
[0268] According to non-reducing SDS-PAGE, PEGylation of anti-ACTH
antibody at cysteine residues did not affect the quaternary
structure of the antibody. In reducing SDS-PAGE of the conjugates
bands at 66 and 75 kDa were detected, which were assigned to mono-
and di-PEGylated heavy chain respectively. PEGylated light chain
was not observed by SDS-PAGE.
Example 5
[0269] Introduction of Thiol Groups Using N-Succinimidyl
S-Acetylthioacetate (SATA) and Subsequent Modification with
PEG-Alkene
[0270] According to SEC analysis the quaternary structure of the
antibodies was not affected by partial reduction and conjugation of
PEG to the respective cysteine residues. However reduction of
disulfide bonds might weaken the antibody stability and interfere
with its activity. Therefore, introduction of free thiol groups by
use of the bi-functional reagent SATA should be investigated. By
this approach SATA is initially attached via its NHS function to
free amino groups of lysine residues in the antibody. Subsequently
the sulfhydryl group of the conjugated SATA is de-protected by
treatment with hydroxylamine:
##STR00012##
[0271] Deprotection of Sulfhydryl Group by Treatment with
Hydroxylamine:
##STR00013##
[0272] The resulting free thiol group should be conjugated with
PEG-alkene/-alkyne by photo reaction under previously developed
conditions.
5.1. Experimental
[0273] Attachment of SATA to Free Amino Groups of Antibodies--
[0274] For attachment of SATA to IgG1 and anti-ACTH antibody the
SATA and Sulfhydryl Addition Kit (Thermo Scientific) was used. The
reaction was performed based on the manufacturer's instructions.
Antibody at a concentration of 1 mg/mL in 20 mM Na phosphate pH 7.2
was modified with SATA at molar excesses of 1, 3, 5 and 10
equivalents. The reaction was performed for 2 hrs. at room
temperature. The sulfhydryl group of the conjugated SATA was
deprotected by treatment with hydroxylamine at a concentration of 5
mg/mL for 2 h at room temperature. Residual free SATA and
hydroxylamine were removed using CentriSpin 10 columns.
[0275] Conjugation of PEG-Alkene/-Alkyne to Free SATA Thiol
Groups--
[0276] PEG-alkene/-alkyne was attached to the free thiol group of
SATA conjugated to the antibodies by photo induction using a
CAPROBOX.TM. (Caprotech). The reaction was performed at a protein
concentration of 1 mg/mL and a 10-fold PEG-alkene/-alkyne excess in
20 mM Na phosphate pH 7.2. 1 mole photoinitiator I per mole PEG was
added and a light exposure time of 30 s was chosen.
5.2 Results
[0277] In initial experiments, attachment of PEG-alkene to free
thiol groups of SATA molecules attached to amino groups of antibody
IgG1 was investigated using different SATA excesses. The resulting
PEGylation reaction mixtures were analyzed by SDS-PAGE
[0278] In non-reducing SDS-PAGE a main band at 155 kDa
corresponding to the whole IgG1 was detected. Additional bands at
lower molecular weight (approx. 135 and 110 kDa) were assigned to
partially dissociated IgG1. The ratio of these bands increased with
higher SATA excesses, which might indicate that attachment of SATA
affected the quaternary structure of the antibody. Therefore the
excess of SATA over antibody should be minimized. In reducing
SDS-PAGE a band at 66 kDa which amounted to 1-2% of the total
protein corresponded to the mono-PEGylated heavy chain. Additional
bands at higher molecular weight were assigned to high molecular
weight impurities (HMWI), which might correspond to aggregates or
multiply PEGylated protein. For further investigation, a control
reaction was performed, for which IgG1 was modified with 3 or 10
eq. SATA. Subsequently the photo click reaction was performed under
standard conditions without addition of PEG-alkene.
[0279] In reducing SDS-PAGE bands referring to HMWI as observed in
the PEGylation reactions were detected. As no PEG was added to the
mixtures during the photo click reaction, these bands were assigned
to aggregated protein.
[0280] In subsequent experiments attachment of PEG-alkene to SATA
conjugated with anti-ACTH antibody at 1- and 3-fold excess by photo
click chemistry was investigated.
[0281] Using anti-ACTH antibody, a lower ratio of aggregates was
observed after the photo click reaction compared to IgG1. A band
corresponding to the mono-PEGylated heavy chain of anti-ACTH
antibody was detected by reducing SDS-PAGE analysis. Thus
PEGylation of the antibody is considered feasible, however, the
ratio of mono-PEG-HC was low (<1%).
Example 6
[0282] Surface Modification of Gold Surface with Alkene/Alkyne and
Covalent Coupling of Proteins (Antibodies) Via Photo-Reaction.
6.1. Equipment
[0283] The equipment listed in the following table is exemplary.
Equipment of comparable quality from other manufacturers may be
used instead.
TABLE-US-00002 Description, type Manufacturer (Country) UV-Lamp UV3
Thermo-Fisher (D) 0.2 mL cups VWR (D) 1.5 mL cups VWR (D)
Thermomixer comfort Eppendorf (D) Vortex shaker Neolab (D) pH-meter
Innolab 1 WTW (D) 1000 .mu.L, pipette Eppendorf (D) 2-100 .mu.L
pipette Eppendorf (D) 0.5-10 .mu.L pipette Eppendorf (D) 15 mL
screw cap tubes, PE sterile Sarstedt (D) Spotting Device S11
Scienion (D)
6.2. Materials
[0284] The materials listed in the following table are exemplary.
Materials of comparable quality from other manufacturers may be
used instead.
TABLE-US-00003 Name/Abbreviation, common Quality/ Provider/ name
Specification Manufacturer PEG-alkene/-alkyne (F)/(G) Examples
1.4./1.5. Antibody Distinct supplier Sodium dihydrogen phosphate
Ph. Eur. E. Merck (DE) monohydrate
(NaH.sub.2PO.sub.4.cndot.H.sub.2O), Disodium hydrogen phosphate Ph.
Eur. E. Merck (DE) (Na.sub.2HPO.sub.4) Water, Rectapur Deionized
ProLabo/VWR (DE) water TCEP, Tris(2- Powder, .gtoreq.98%
Sigma-Aldrich (DE) carboxyethyl)phosphine hydrochloride
Photoinitiator, 2-hydroxy-4-(2- >98.0% TCI Deutschland
hydroxyethoxy)-2- (HPLC) GmbH (DE) metylpropiophenone DMAC,
N,N-Dimethylacetamide 99.9% (GC) Sigma-Aldrich (DE)
6.3. Preparation of Solutions and Buffers
[0285] 10 mM Sodium Phosphate Buffer pH 7.2 [0286] Weigh 710 mg of
disodium hydrogen phosphate in a 250 mL beaker and fill up with 250
mL ddH.sub.2O; weigh 780 mg of sodium dihydrogen phosphate into
another 250 mL beaker and fill up with ddH.sub.2O [0287] Fill 250
mL disodium hydrogen phosphate in a 500 mL beaker and titer with
dihydrogen phosphate until pH 7.2 is reached. [0288] Filter the
solution into a 500 mL glass bottle, using a 0.2 .mu.m polyether
sulfone membrane filter. [0289] Store the solution at room
temperature for up to six months.
[0290] 25 mg/mL TCEP Stock Solution in ddH.sub.2O [0291] Weigh 25
mg TCEP into a 1.5 mL screw cap tube. [0292] Add 1 mL of ddH.sub.2O
[0293] Vortex the tube until the TCEP is completely dissolved.
[0294] Store at 4.degree. C.
[0295] 1 mg/mL Photoinitiator in 20 mM Sodium Phosphate Buffer, pH
7.2 [0296] Weigh 10 mg photoinitiator into a 15 mL screw cap tube.
[0297] Add 10 mL of 10 mM sodium phosphate buffer, pH 7.2. [0298]
Shake the solution 10 min at 40.degree. C. to dissolve the
photoinitiator. [0299] Pass the solution through a 0.2 .mu.m
polyether sulfone syringe filter. [0300] Prepare the solution on
the day of use.
[0301] 10 mM Solution of PEG-Alkene/-Alkyne in DMAC and Gold
Surface Modification [0302] PEG-alkene/-alkyne is stored at
-20.degree. C.; thaw at room temperature for approx. 30 min. [0303]
Weigh appropriate mass PEG-alkene/-alkyne into a 15 mL screw cap
tube (5000 Da PEG-Alkene.fwdarw.dissolve 500 mg in 1 mL DMAC to
obtain 100 mM stock solution). [0304] Vortex the PEG solution until
the PEG is completely dissolved. [0305] 100 mM stock solution can
be aliquoted and stored at -20.degree. C. [0306] Prepare working
solution on the day of use therefore: [0307] Dilute stock solution
with DMAC to 10 mM working solution concentration. [0308] Invert
and vortex the solution to homogenize. [0309] Pipette 10 .mu.L 10
mM PEG-alkene/alkyne solution to cleaned (wash protocol) gold CMOS
surface and let react for 60 min 20.degree. C. in a glass petri
dish within a fume hood. [0310] Wash gold CMOS surface with
ddH.sub.2O within a 250 mL beaker for 10 s. [0311] Dry gold CMOS
surface with pressurized air. [0312] Store dry until use at
20.degree. C.
[0313] TCEP Reduction of Disulfide Bonds [0314] Pipette 20 .mu.L
Antibody (0.2 mg/mL, MW=150 kDa) into a 0.5 mL Cup. [0315] Dilute
the TCEP stock solution 1:100 two times and 1:2 with sodium
phosphate buffer, to give a final TCEP concentration of 3.times.
molar excess related to antibody concentration [0316] Add 20 .mu.L
TCEP to the antibody [0317] Incubate the reduction mixture 60 min
at room temperature and agitation at 30 rpm.
[0318] Spotting and Coupling of Antibodies [0319] Pipette 15 .mu.L
Spotting-buffer into a Genetix Plate [0320] Add 2.24 .mu.L of
photoinitiator stock solution [0321] Add 12.76 .mu.L reduced
antibody solution two times higher concentrated than spotting
concentration [0322] Spotting (standard spotting procedure) [0323]
Place the UV-Lamp in the Spotting device and expose to UV-light
(.lamda.=302 nm) for at least 4 min-up to 10 min with a distance of
10 mm above gold surface. [0324] Wash with ddH.sub.2O and dry with
pressurized air [0325] Block surface with desired blocking agent
(blocking protocol) [0326] Wash with ddH.sub.2O and dry with
pressurized air [0327] Use CMOS for intended assay
[0328] The upper part of FIG. 4 shows schematically the metal
surface (1), which is modified by the cross-linker molecules (4)
being covalently linked to the metal surface via the dithiol group,
having a polyethylene glycol (PEG) group as spacer and a terminal
allyl group. Upon adding a biomolecule, which has at least one
sulfhydryl group (2), a photo-initiator and irradiation with UV
light, the biomolecule is immobilized (3) on the surface via the
cross-linker as shown in the lower part of FIG. 4.
Examples 7 to 9
[0329] The results of the different immobilization experiments are
shown in the fluorescence pictures of FIGS. 1 to 3, which have been
made, in order to assess the surface modification of CMOS
chips.
[0330] The CMOS chips comprise 128 electrodes, each of which can be
addressed via spotting (cp. Example 6). The corresponding spotting
layouts are also shown.
Example 7
[0331] Influence of UV light and photo initiator in comparison with
a lipoamide-PEG(11)-maleimide modified surface.
[0332] In the following experiment, fluorescence pictures have been
taken from a CMOS chip, which consist of 128 gold-electrodes as
sensor surface. Different dilutions and spotting puffer solutions
have been used for spotting a polyclonal rabbit anti-ACTH antibody.
Subsequently, a complete assay for ACTH has been carried out,
adding ACTH to the surface. A monoclonal mouse anti-ACTH antibody
binds after the addition to bound ACTH and can be visualized with
fluorescence marked rabbit anti-mouse antibody.
[0333] The intensity of the fluorescence is directly proportional
to the amount of the bound polyclonal rabbit anti-ACTH antibody. In
FIG. 1 the different conditions of the photo reaction in comparison
to a lipoamide-PEG(11)-maleimide modified surface are shown. [0334]
A: A lipoamide-PEG(11)-maleimide modified surface with 10 min UV
irradiation at 304 nm wavelength. Only a weak occurrence of
fluorescence can be observed. This means that the immobilization
reaction of the antibody had taken place only to a low degree.
[0335] B: An R-.alpha.-lipoic-acid-PEG12-propargyl modified surface
according to the invention with 7.5 min irradiation time. It is
apparent that an immobilization of the antibody must have taken
place, due to the higher fluorescence intensities. [0336] It is
noteworthy that no photo initiator had been used in the samples
KIA5-KIA7, whereas samples KIA1-KIA4 were generated with photo
initiator. All antibodies were previously treated with TCEP,
wherein different excesses of TCEP (30.times., 3.times. molar
excess with respect to the concentration of the antibody) have been
employed. [0337] C: An R-.alpha.-lipoic-acid-PEG12-propargyl
modified surface without UV irradiation. It is apparent that only
little immobilization of the antibody at the surface takes place
without UV irradiation. [0338] D: Spotting-Layout KIA represents
different reaction conditions of the polyclonal rabbit anti-ACTH
antibody. SPO: represents the spotting control, where only the
spotting buffer has been applied.
Example 8
[0339] Immobilization of a Monoclonal Mouse Anti-ACTH Antibody with
and without Photo Initiator
[0340] In this experiment, the applicability of the photoreaction
for the immobilization of monoclonal mouse antibodies has been
demonstrated. The antibodies had been dyed directly with a
fluorescence marked anti-mouse antibody. The corresponding results
are shown in FIG. 2.
[0341] FIG. 2 shows the immobilization of a monoclonal mouse
antibody with the aid of the photoreaction according to the
invention on R-.alpha.-lipoic-acid-PEG12-propargyl modified surface
and 4 min irradiation time. [0342] A: A comparison of the
efficiency of the immobilization with and without use of a photo
initiator is shown. It is apparent, that the use of a
photoinitiator achieves an intense and uniform immobilization of
the monoclonal antibody. However, without photo initiator, the
efficiency of the immobilization is less pronounced and the
formation of zones can be recognized. Nevertheless, it can be
assumed, that the reaction can also be conducted without photo
initiator at sufficient irradiation time. [0343] B: The spotting
layout KIA corresponds to a polyclonal rabbit anti-ACTH antibody,
which cannot be dyed. SPO: represents the spotting control, where
only the spotting buffer has been applied. MIA: represents the
monoclonal mouse antibody, wherein MIA1 MIA2 have been generated
with and without photo initiator. All antibodies have been treated
with TCEP previously.
Example 9
[0344] Comparison of Allyl and Propargyl Functionalities as Well as
Defined and Non-Defined PEG Chain Lengths.
[0345] In this experiment, the reactivity of allyl and propargyl
groups has been compared. In addition, the difference of the
reactivity of defined and non-defined PEG chain lengths has been
investigated. Non-defined PEG chain lengths are those having an
average molecular mass of 5 kDa. The assays have been carried out
as described in example 7 with ACTH as analyte. The utilized CMOS
chips have been pre-treated with a layer of benzocyclobutene (BCB),
whereupon the interspaces between the electrodes show enhanced
fluorescence. The antibodies have been applied in a 1:2 dilution
series starting at 100 .mu.g/mL. The corresponding results are
shown in FIG. 3. Irradiation time was 4 min.
[0346] FIG. 3 shows the immobilization of a polyclonal rabbit
anti-ACTH antibody with the photoreaction according to the
invention on auf different modified surfaces. [0347] A:
R-.alpha.-lipoic-acid-5 kDa PEG-propargyl (Example 1.5) modified
surface. [0348] B R-.alpha.-lipoic-acid-PEG12-propargyl (Example
1.2) modified surface. [0349] C: R-.alpha.-lipoic-acid-5 kDa
PEG-allyl (Example 1.4) modified surface. [0350] D:
R-.alpha.-lipoic-acid-PEG12-allyl (Example 1.1) modified surface.
[0351] E: The spotting-layout KIA corresponds with a polyclonal
rabbit anti-ACTH antibody, wherein KIA shows the highest
concentration (100 .mu.g/mL as spotting solution), whereas KIA4
contains the lowest concentration (12.5 .mu.g/mL as spotting
solution). It is apparent, that D shows the highest intensity of
fluorescence. All the other modified surfaces show slightly lower
intensities, but a specific immobilization can be noted here,
too.
* * * * *